Tesis sobre el tema "Aldosteronism"

Red blood cells (RBCs) are a-nucleated cells particularly exposed to different stimuli, among which circulating hormones and intra/extra-cellular derivatives from oxidization processes. In addition, our previous studies showed that in the case of inflammatory diseases with GSH content alterations, RBCs were much more sensitive to diamide, a mild oxidant able to trigger tyrosine-phosphorylation (Tyr-P) of membrane proteins, mainly band 3. Aldosterone (Aldo), mineralocorticoid hormone, has been shown to induce many effects other than the common diuretic process regulation and involving the expression and activation of the superoxide generating enzyme NADPH oxidase, thus potentially explaining the increased plasma markers of oxidative stress (OS) like isoprostanes in primary aldosteronism (PA), disease characterized by excessive Aldo secretion. This well-known Aldo action is mediated by the activation of a cytosolic specific receptor, the mineralocorticoid receptor (MR), in the so called genomic pathway, which distinguishes from the direct effect of Aldo on many proteins and enzymes in the second mechanism, also known as non-genomic pathway. Starting from these evidences, if a direct Aldo involvement in the triggering of inflammatory-related oxidative status of the cells was evolving, RBCs were the eligible cells for the investigation of the non-genomic Aldo pathway. The study involved PA patients and healthy control (HC) and consisted with three phases: i) a first approach was carried out to evidence potential alterations in PA RBCs followed by an in vitro deepening to assess the effective direct/indirect involvement of Aldo in these alterations; ii) once identified as responsible of the RBCs alterations found in PA, Aldo pathway within HC RBCs cytosol was investigated; iii) at last, Aldo-related pathway in RBCs was studied with particular attention to the mechanism leading to membrane band 3 alterations starting from Aldo induced receptor activation. In the first part, PA RBCs were showed to have oxidative-like alterations such as band 3 protein increase of both Tyr-P level and clustering, thus suggesting that PA could be linked to other inflammatory diseases. The effects of Aldo, cortisol (Cort) and canrenone (Can) (added as agonist and inhibitor, respectively) were compared and Aldo was confirmed as the only responsible of the alterations previously observed in PA RBCs. Furthermore, Aldo was shown to trigger RBCs membrane alterations leading to autologous IgG binding in a sort of premature ageing of the cells. The second part of the study analyzed the mechanism of Aldo action: for the first time MR was identified in RBCs cytosol as a soluble multi-protein complex, differently regulated by the effector utilized. In facts, in the presence of Aldo, MR broke away from the complex to form dimers which were promptly proteolysed in a sort of turn off signaling. Can or Cort were not able to trigger similar events, thus explaining the different alterations found on RBCs membranes. However, since to now no direct evidence was found about the possibility that Aldo induced an increase of oxidation status in RBCs, oxidization level in both membranes and cytosol was addressed in the third and last part of the study. Results showed no difference in GSH and GSSP contents and carbonic anhydrase (CA) monomerization and activity between HC and PA RBCs. In contrast, preliminary data would confirm a sort of oxidative-related increase of band 3 disulfide bond formation, thus suggesting a new intriguing mechanism leading to band 3 increased oxidative status without changing the common anti-oxidative cellular defenses. Further investigations addressing this mechanism are in progress. In conclusion, we found that in PA RBCs Aldo is responsible for the membrane alterations leading to a potential premature removal of the cells from circulation. Aldo exerts its effect through the activation of the soluble MR complex, which participates in the modulation of the Aldo signaling through the possibility of being differently affected by other steroids or Aldo inhibitors (Can). Further studies are in progress to explore both nature and potential mediators of the Aldo-induced alterations in the band 3 dimer formation.
Gli eritrociti (RBCs) sono cellule non nucleate particolarmente esposte a differenti stimoli, tra i quali l’effetto degli ormoni circolanti nel sangue e i derivati dei processi di ossidazione intra o extracellulari. Studi precedenti condotti nel nostro laboratorio hanno dimostrato che, nel caso di malattie infiammatorie con alterazione del contenuto di GSH rispetto ai controlli, gli eritrociti erano molto più sensibili alla diamide, un blando ossidante in grado di innescare la tirosin-fosforilazione (Tyr-P) delle proteine di membrana, principalmente della proteina banda 3. L’aldosterone (Aldo), ormone mineralocorticoide, oltre alla sua classica azione regolatoria dei processi diuretici, è in grado di indurre molti altri effetti tra i quali l’espressione e l’attivazione dell’enzima NADPH ossidasi, generatore di anione superossido. Questo fatto potrebbe potenzialmente spiegare l’incremento di marker plasmatici di stress ossidativo (OS) come gli isoprostani, nell’aldosteronismo primitivo (PA), patologia caratterizzata da un’eccessiva secrezione di Aldo. Partendo da queste evidenze, gli RBCs erano cellule ottimali per studiare se l’Aldo potesse indurre un aumentato stato di ossidazione determinato da una sua azione diretta sui processi infiammatori. Infatti, in queste cellule non nucleate, un eventuale coinvolgimento dell’Aldo nei meccanismi infiammatori, mediante un’azione non-genomica, sarebbe stato univocamente dimostrato. Lo studio ha coinvolto sia pazienti con PA che controlli sani (HC) e si è svolto in tre fasi: i) in un approccio iniziale abbiamo valutato se esistessero potenziali alterazioni negli RBCs dei pazienti, procedendo, poi, con un approfondimento in vitro condotto sugli RBCs di HC per confermare o meno un diretto coinvolgimento dell’Aldo nell’indurre queste alterazioni; ii) una volta identificato come responsabile effettivo delle alterazioni riscontrate, abbiamo cercato di chiarire il meccanismo di azione dell’Aldo a livello citosolico; iii) infine, partendo dall’evidenza che l’azione dell’Aldo veniva mediata dall’attivazione del recettore citosolico, abbiamo cercato di capire il meccanismo attraverso cui questa attivazione si trasmettesse, a livello delle membrane. Nella prima parte, abbiano dimostrato che negli RBCs dei pazienti erano presenti delle alterazioni quali un incremento sia della Tyr-P della banda 3 che una sua aggregazione, suggerendo, così, che la patologia potesse essere correlata ad uno stress ossidativo come dimostrato in altre malattie infiammatorie. Inoltre, dopo aver comparato gli effetti di Aldo, cortisolo (Cort) e canrenone (Can) (aggiunti rispettivamente come agonista ed inibitore), abbiamo confermato che era proprio l’Aldo il diretto responsabile delle alterazione che, in ultima, portavano ad un aumento della quantità degli anticorpi autologhi legati alla membrana, rispecchiando una prematuro invecchiamento cellulare. Nella seconda parte dello studio, abbiamo dimostrato, per la prima volta, la presenza a livello citosolico del recettore dei mineralocorticoidi (MR), che risulta essere presente in un complesso multi-proteico di elevato peso molecolare. Inoltre, abbiamo evidenziato come solo l’Aldo inducesse la liberazione dell’MR dal complesso a formare dimeri prontamente proteolizzati in una sorta di spegnimento del segnale. Al contrario, né Cort né Can erano in grado di indurre l’attivazione del recettore. Tuttavia, poiché finora non è mai stato dimostrato se l’Aldo potesse indurre un aumento delle stato ossidativo dell’eritrocita, nella terza parte dello studio abbiamo analizzato alcuni markers di ossidazione sia a livello di membrana che di citosol. I nostri risultati indicano che nessuna modifica del contenuto di GSH o di proteine glutationilate (GSSP) era presente nei pazienti rispetto ai controlli, come nessuna alterazione nella monomerizzazione e attivazione della anidrasi carbonica (CA), nuovo parametro nella valutazione di un aumentato stato di ossidazione. Tuttavia, i nostri risultati mostrano che la proteina banda 3 risulta effettivamente sottoposta ad uno stress ossidativo che ne induce l’aggregazione attraverso la formazione di ponti disolfuro. Risultato, questo, che merita ulteriori indagini ed approfondimenti. In conclusione, abbiamo trovato che negli RBCs dei pazienti con PA l’Aldo è responsabile di alterazioni di membrana che portano ad una potenziale prematura rimozione delle cellule dalla circolazione. L’azione dell’Aldo viene mediata a livello citosolico dall’MR, ma non dal Cort. Ulteriori studi sono in corso per esplorare sia la natura che il meccanismo di potenziali mediatori dell’effetto dell’Aldo-MR a livello delle membrane eritrocitarie.

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.
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.

After the elucidation of the underlying genetic cause in many Mendellian diseases molecular medicine has turned its attention to the more common polygenic disorders. High blood pressure is one such complex trait which has been the subject of much interest in recent years. A number of candidate loci have been found to different groups to influence blood pressure and have been implicated in the aetiology of essential hypertension although the most significant discoveries have been in the single gene causes of high blood pressure. Glucocorticoid remediable aldosteronism (GRA) is an autosomal dominant cause of high blood pressure and primary aldosteronism caused by a chimaeric 11- hydroxylase/aldosterone synthase gene. The presence of this chimaeric gene causes aldosterone to be produced in response to adrenocorticotrophic hormone (ACTH) which results in primary aldosteronism and hypertension. GRA is characterised by the normalisation of both blood pressure and serum biochemistry by the administration of dexamethasone. Following screening of 356 individuals from the hypertension clinic in Aberdeen we identified 5 families with the chimaeric gene characteristic of GRA. These patients were initially diagnosed on the basis of an RFLP for the restriction endonuclease BamHI. We show that a long PCR technique can be used reliably to identify patients with the chimaeric gene and that cloning and sequencing of the chimaeric long PCR product can identify the point of crossing over in these families. Members of each of the 5 kindreds were then tested for some of the other candidate gene polymorphisms implicated in the aetiology of essential hypertension to investigate the possibility that these variants could influence the level of blood pressure in GRA patients.

Primary aldosteronism (PA) is a common form of secondary hypertension with an international prevalence rates between 5 and 10 %. It is characterized by a high autonomous aldosterone production that causes cardiovascular damage, renin suppression, hypertension, sodium retention, potassium excretion and hypokalemia. The screening of PA is a simple test measuring aldosterone to renin ratio (ARR) with immunoassay method. This test is currently considered as the most reliable screening tool for PA.     The main objective of the study was to evaluate an ELISA-method, for detection of aldosterone and renin in blood plasma, to be used for routine analysis in the laboratory. The second aim was to investigate the effect of refreezing samples, considering that cryoactivation of prorenin might occur.     One hundred blood samples were analysed, in regard to aldosterone and renin, by using two commercial ELISA assays (DRG ELISA from DRG Diagnostics, Germany) on a Dynex DS2 instrument. In addition, the accuracy and precision of the methods were calculated. The effect of refreezing was investigated with a series of eight samples, which were analyzed twice on the same instrument.     Both assays performed well. The resulting data showed good precision and accuracy. The correlation between the original and refreezed samples was good, r = 0.989 and r = 1.0 for aldosterone and renin respectively. Considering that the study only included eight samples, further investigation is recommended.     Evaluation showed that both immunoassays are reliable in diagnostic use and the ELISA-method is suitable to implement in the laboratory for routine analysis.

Background Primary aldosteronism (PA) is the most common cause of arterial hypertension characterized by high levels of aldosterone, resulting in excessive mineralocorticoid receptor (MR) stimulation, and extensive hypertensive-mediated organ damage (HMOD). Current guidelines recommend one or more exclusion tests in patients performed the screening test with the measurement of aldosterone-to-renin ratio (ARR) to avoid further lateralization procedures in those who tested false-positive. To date the diagnostic gain provided by these exclusion tests over the ARR was examined only in few studies, and, therefore, stands on a weak level of evidence. Moreover, growing experimental evidence has shown that immune system, especially T cells is involved in aldosterone-induced HOMD through MR activation. MR activation in animal models of hyperaldosteronism promoted T cells differentiation to the pro-inflammatory T helper 17 (Th17) subsets while decreasing the number of anti-inflammatory T regulatory (Tregs). Alteration of the balance between Th17 and Tregs contributed to the pathogenesis of hypertension and the associated complications. Furthermore, our previous work provided proof on the MR gene expression and protein expression in both human CD4+ and CD8+ T cells by Droplet Digital PCR and immunoblotting, respectively. However, up to now, there was no relevant research focused on the function of Th17 and Tregs in PA patients, and evaluate the effect of MR antagonists and surgery on these cells in patients with PA. Aims - To meta-analyze available studies of exclusion tests to furnish a more accurate picture of their diagnostic accuracy and gain in the work-up of PA with a higher level of confidence. - To investigate the levels of circulating Th17 and Tregs in PA patients and evaluate the effect of MR antagonists and surgery on these cells in PA patients. Materials and methods - Eligible studies reported on the diagnostic performance of the ARR and the exclusion tests for identifying unilateral PA (uPA) were selected using the “gold” standard (biochemical cure after adrenalectomy), or, whenever unavailable, a “golden” standard (adrenal imaging and/or AVS) as reference. Then, pooled sensitivity, specificity, the summary receiver operating characteristic (sROC) curve, and corresponding area under the curve (sAUC) were examined. - Blood samples from PA patients were obtained at 3-time points: before surgery, when patients had high PAC and were not treated with MR antagonists (T0); before surgery when patients had high PAC and were treated with MR antagonists (T1); one month after surgery when patients had normal PAC (T2). Immunologic markers on Th17 (CD4+IL17+), pathogenic IL-23-dependent Th17 (CD4+IL17+IL23R+), and Tregs (CD4+CD25+FoxP3+) were analyzed by multicolor flow cytometry. Results - By increasing the overall sample size of the patients studied by these tests and comprising the experience gained in multiple centers, we found that two most popluar exclusion tests, captopril challenge test (CCT) and saline infusion test (SIT), had no diagnostic gain over ARR for diagnosing uPA. - The percentage of circulating Th17 in PA patients was significantly lower after treatment of MR antagonists and post-surgery biochemical cure; meanwhile, there was a decrease in pathogenic Th17 one month after surgery. Although there were no differences in the percentage of Tregs at these 3-time points, Th17/Tregs ratio was markedly decreased after treatment with MR antagonists and post-surgically cure of the hyperaldosteronism. Conclusions This meta-analysis revealed that the use of exclusion tests in patients with a high post-test probability of uPA, as identified by ARR values, could be unnecessary, if not confounding. Meanwhile, our current study showed that treatment with MR antagonists and post-surgery biochemical cure can decrease the percentage of circulating Th17 and the ratio of Th17/Tregs in PA patients.
Background Primary aldosteronism (PA) is the most common cause of arterial hypertension characterized by high levels of aldosterone, resulting in excessive mineralocorticoid receptor (MR) stimulation, and extensive hypertensive-mediated organ damage (HMOD). Current guidelines recommend one or more exclusion tests in patients performed the screening test with the measurement of aldosterone-to-renin ratio (ARR) to avoid further lateralization procedures in those who tested false-positive. To date the diagnostic gain provided by these exclusion tests over the ARR was examined only in few studies, and, therefore, stands on a weak level of evidence. Moreover, growing experimental evidence has shown that immune system, especially T cells is involved in aldosterone-induced HOMD through MR activation. MR activation in animal models of hyperaldosteronism promoted T cells differentiation to the pro-inflammatory T helper 17 (Th17) subsets while decreasing the number of anti-inflammatory T regulatory (Tregs). Alteration of the balance between Th17 and Tregs contributed to the pathogenesis of hypertension and the associated complications. Furthermore, our previous work provided proof on the MR gene expression and protein expression in both human CD4+ and CD8+ T cells by Droplet Digital PCR and immunoblotting, respectively. However, up to now, there was no relevant research focused on the function of Th17 and Tregs in PA patients, and evaluate the effect of MR antagonists and surgery on these cells in patients with PA. Aims - To meta-analyze available studies of exclusion tests to furnish a more accurate picture of their diagnostic accuracy and gain in the work-up of PA with a higher level of confidence. - To investigate the levels of circulating Th17 and Tregs in PA patients and evaluate the effect of MR antagonists and surgery on these cells in PA patients. Materials and methods - Eligible studies reported on the diagnostic performance of the ARR and the exclusion tests for identifying unilateral PA (uPA) were selected using the “gold” standard (biochemical cure after adrenalectomy), or, whenever unavailable, a “golden” standard (adrenal imaging and/or AVS) as reference. Then, pooled sensitivity, specificity, the summary receiver operating characteristic (sROC) curve, and corresponding area under the curve (sAUC) were examined. - Blood samples from PA patients were obtained at 3-time points: before surgery, when patients had high PAC and were not treated with MR antagonists (T0); before surgery when patients had high PAC and were treated with MR antagonists (T1); one month after surgery when patients had normal PAC (T2). Immunologic markers on Th17 (CD4+IL17+), pathogenic IL-23-dependent Th17 (CD4+IL17+IL23R+), and Tregs (CD4+CD25+FoxP3+) were analyzed by multicolor flow cytometry. Results - By increasing the overall sample size of the patients studied by these tests and comprising the experience gained in multiple centers, we found that two most popluar exclusion tests, captopril challenge test (CCT) and saline infusion test (SIT), had no diagnostic gain over ARR for diagnosing uPA. - The percentage of circulating Th17 in PA patients was significantly lower after treatment of MR antagonists and post-surgery biochemical cure; meanwhile, there was a decrease in pathogenic Th17 one month after surgery. Although there were no differences in the percentage of Tregs at these 3-time points, Th17/Tregs ratio was markedly decreased after treatment with MR antagonists and post-surgically cure of the hyperaldosteronism. Conclusions This meta-analysis revealed that the use of exclusion tests in patients with a high post-test probability of uPA, as identified by ARR values, could be unnecessary, if not confounding. Meanwhile, our current study showed that treatment with MR antagonists and post-surgery biochemical cure can decrease the percentage of circulating Th17 and the ratio of Th17/Tregs in PA patients.

Background. Previous studies reported that primary aldosteronism (PA) is associated with an increased prevalence of anxiety, depression and subnormal quality of life (QoL) scores that may be improved after surgical treatment. Aim of the Study. The aim of the study was to assess the impact of surgery on health-related QoL and depression status of patients suffering from PA, comparing the results with a control group of patients undergoing surgery for a non-secreting adrenal tumor. Materials and Methods. Data on QoL and depression status were prospectively collected, from January 2014 to October 2016, before, early after surgery (at 1 month) and at long term (at least 6 months) in patients with unilateral PA and in a control group of patients with non-secreting adrenal tumor submitted to unilateral transperitoneal laparoscopic adrenalectomy. QoL was assessed using the Short Form 36 (SF-36) Health Survey for Physical (PCS) and Mental Component (MCS); the depression status by a 20-item depression scale (DS) questionnaire. Results. Twenty-six PA patients and 15 controls were recruited. Biochemical cure of the disease was achieved following surgery in all PA patients; hypertension was cured in 31% of cases and improved in the remaining 69% of cases. No morbidity occurred in both groups. There were no significant differences between PA patients and controls concerning demographics, preoperative PCS, MCS and DS values. In patients with PA, MCS values improved at early (42.72±13.68 vs 51.56±9.03, p=0.0005) and long term follow up (42.72±13.68 vs 51.81±7.04, p<0.0001); also DS values improved at early (15.92±11.98 vs 8.3±8.8, p=0.0002) and long term follow up (15.92±11.98 vs 4.57± 6.11, p<0.0001). In PA patients PCS values significantly improved at long term follow up (51.02±8.04 vs 55.85±5.1, p=0.013). Also in controls an improvement of MCS and DS scores was found at early and long term follow up compared to preoperative values, while no significant differences in PCS were found. Conclusions. Both PA and non-secreting adrenal tumors affect health-related QoL, worsening MCS and DS scores. Adrenalectomy is effective in curing PA, and improving MCS and DS scores at early and long-term follow-up, in patients with PA and non-secreting adrenal tumors. In PA patient surgery also significantly improves PCS at long term follow up.
Presupposti dello studio. Studi precedenti hanno descritto che l’iperaldosteronismo primario (PA) è associato ad un aumento della prevalenza di ansia, depressione e peggioramento della qualità di vita (QoL), con miglioramento significativo dopo il trattamento chirurgico. Scopo dello studio. Lo scopo dello studio è quello di indagare l’impatto della chirurgia sulla qualità di vita dei pazienti con PA, comparando i risultati con un gruppo di controllo costituito da pazienti sottoposti a surrenectomia per tumori surrenalici non secernenti. Materiali e Metodi. I dati sulla qualità di vita e lo stato di depressione sono stati raccolti prospetticamente, da Gennaio 2014 a Ottobre 2016, preoperatoriamente, dopo 1 mese e dopo almeno 6 mesi dall’intervento chirurgico di surrenectomia laparoscopica transperitoneale in pazienti con PA lateralizzato e in un gruppo di controllo costituito da pazienti con tumori non-secernenti del surrene. La QoL è stata valutata utilizzando il questionario SF-36, per valutazione della componente fisica (PCS) e mentale (MCS); lo stato depressivo è stato quantificato utilizzando un questionario di valutazione della depressione (DS) costituito da 20 domande. Risultati. Sono stati reclutati 26 pazienti con PA e 15 controlli. La cura biochimica della malattia è stata ottenuta in tutti i pazienti con PA; l'ipertensione è stata curata nel 31% dei casi ed è migliorata nel 69% dei casi. Non si sono verificate complicanze chirurgiche in entrambi i gruppi. Non sono state rilevate differenze statisticamente significative tra i pazienti con PA e i controlli per quanto riguarda i dati demografici e i valori preoperatori di PCS, MCS e DS. Nei pazienti con PA, i valori di MCS sono migliorati sia a 1 mese dall’intervento (42.72 ± 13.68 vs 51.56 ± 9.03, p = 0,0005) che a distanza (42,72 ± 13.68 vs 51.81 ± 7.04, p <0,0001). Nei pazienti affetti da PA anche i valori di DS sono migliorati sia a breve (15.92 ± 11.98 vs 8.3 ± 8.8, p = 0,0002) che a lungo termine (15,92 ± 11.98 vs 4.57 ± 6.11, p <0,0001); i valori di PCS sono migliorati significativamente solo a distanza dall’intervento (51.02 ± 8.04 vs 55.85 ± 5.1, p = 0,013). Anche nel gruppo di controllo i valori di MCS e DS sono migliorati sia a breve dopo l’intervento che a distanza; in questi pazienti non sono state rilevate variazioni significative nei valori di PCS dopo l’intervento. Conclusioni. I pazienti affetti da PA e tumori surrenalici non-secernenti sono caratterizzati da una QoL peggiore rispetto alla popolazione normale. La surrenectomia è efficace nella cura del PA, e nel migliorare i punteggi di MCS e DS sia a 1 mese dall’intervento che a lungo termine, sia nei pazienti con PA che nel gruppo di controllo. Nei pazienti con PA, i valor di PCS migliorano significativamente solo a lungo termine.

In Primary Aldosteronism (PAL) excessive amounts of aldosterone cause sodium and water retention and, in many individuals, this leads to moderate to severely high blood pressure. Although the chemistry and physiology are increasingly well understood, including the outcomes of treatment on physical health, there has been no systematic study of the psychological dimension of PAL. Anecdotally, patients exhibit symptoms such as angry outbursts, irritability, anxiety and defensiveness, and partners of these patients sometimes mention poor anger control and brittle or unpredictable moods. This thesis reports a systematic study of anger and anxiety among patients undergoing treatment for PAL. Eighty-three patients were recruited over an 11-month period to a prospective, pre-post design study to determine if treatment was associated with change in psychological state. Participants completed the State-Trait Anger Expression Inventory (STAXI-2), State-Trait Anxiety Inventory (STAI) and Psychosocial Adjustment to Illness Scale (PAIS) questionnaires. Adrenal Vein Sampling confirmed overproduction of aldosterone in one or both adrenal glands. Patients with Aldosterone Producing Adenoma (APA) were offered adrenalectomy. As per usual treatment protocols, patients with Bilateral Adrenal Hyperplasia (BAH) were prescribed spironolactone or amiloride depending predominantly on severity of blood pressure and potassium levels. Post-test questionnaires were completed after 6-8 months. Analysis was by mixed design (between-within subjects) ANOVA. Participant numbers in the adrenalectomy group fell far short of expectations. Fourteen past patients who had undergone unilateral adrenalectomy completed a retrospective semi-structured questionnaire. This qualitative data was analysed to identify themes similar to quantitative data. At baseline, 'non-completers' (ie those who did not complete the post-test; n=19), were significantly more angry than 'completers' (n=50) in State Anger (p< .01), Trait Anger (p< .05) and Anger Expression Index (p< .001). Trait Anxiety was also higher (p< .05), as was Psychological Distress (p< .05). Among those who participated at both interviews, there was small but statistically significant adverse treatment effect with higher scores for State Anger (p< .05), and Feeling Angry (p< .05). However for Trait Anger (p< .01), and 2 of its 3 sub-scales Angry Temperament (p< .05) and Angry Reaction (p< .01) there was a slight to moderate decrease in negative affect with treatment. Psychological Distress scores also improved (p< .05). Across all ANOVAs, there were no significant interaction effects, suggesting that any treatment effect was equivalent for the two drugs. Qualitatively collected data elucidated participants' changes in approach to life and relationships since adrenalectomy. Themes that emerged in the data included improved ability to cope with external stress, better control of emotions, more relaxed relationships and attitude to work, and a greater vitality and quality of life. Generally the comments were consistent with the drug treatments; there was noticeable benefit, including perceived better anger control and less anxiety. Positive psychological effects of treatment observed in the two drug groups were triangulated with data from a qualitative study. The combined evidence suggests that when excess circulating aldosterone is reduced (adrenalectomy), or blocked (spironolactone), or aldosterone's salt and water retaining effects are minimised (amiloride), then nervous irritability and its subsequent psycho-behavioural manifestations are reduced. The effect however is slight and the conclusions are weakened by an apparent attrition bias, and the absence of a control group. Implications for further research are discussed.

In Primary Aldosteronism (PAL) excessive amounts of aldosterone cause sodium and water retention and, in many individuals, this leads to moderate to severely high blood pressure. Although the chemistry and physiology are increasingly well understood, including the outcomes of treatment on physical health, there has been no systematic study of the psychological dimension of PAL. Anecdotally, patients exhibit symptoms such as angry outbursts, irritability, anxiety and defensiveness, and partners of these patients sometimes mention poor anger control and brittle or unpredictable moods. This thesis reports a systematic study of anger and anxiety among patients undergoing treatment for PAL. Eighty-three patients were recruited over an 11-month period to a prospective, pre-post design study to determine if treatment was associated with change in psychological state. Participants completed the State-Trait Anger Expression Inventory (STAXI-2), State-Trait Anxiety Inventory (STAI) and Psychosocial Adjustment to Illness Scale (PAIS) questionnaires. Adrenal Vein Sampling confirmed overproduction of aldosterone in one or both adrenal glands. Patients with Aldosterone Producing Adenoma (APA) were offered adrenalectomy. As per usual treatment protocols, patients with Bilateral Adrenal Hyperplasia (BAH) were prescribed spironolactone or amiloride depending predominantly on severity of blood pressure and potassium levels. Post-test questionnaires were completed after 6-8 months. Analysis was by mixed design (between-within subjects) ANOVA. Participant numbers in the adrenalectomy group fell far short of expectations. Fourteen past patients who had undergone unilateral adrenalectomy completed a retrospective semi-structured questionnaire. This qualitative data was analysed to identify themes similar to quantitative data. At baseline, 'non-completers' (ie those who did not complete the post-test; n=19), were significantly more angry than 'completers' (n=50) in State Anger (p< .01), Trait Anger (p< .05) and Anger Expression Index (p< .001). Trait Anxiety was also higher (p< .05), as was Psychological Distress (p< .05). Among those who participated at both interviews, there was small but statistically significant adverse treatment effect with higher scores for State Anger (p< .05), and Feeling Angry (p< .05). However for Trait Anger (p< .01), and 2 of its 3 sub-scales Angry Temperament (p< .05) and Angry Reaction (p< .01) there was a slight to moderate decrease in negative affect with treatment. Psychological Distress scores also improved (p< .05). Across all ANOVAs, there were no significant interaction effects, suggesting that any treatment effect was equivalent for the two drugs. Qualitatively collected data elucidated participants' changes in approach to life and relationships since adrenalectomy. Themes that emerged in the data included improved ability to cope with external stress, better control of emotions, more relaxed relationships and attitude to work, and a greater vitality and quality of life. Generally the comments were consistent with the drug treatments; there was noticeable benefit, including perceived better anger control and less anxiety. Positive psychological effects of treatment observed in the two drug groups were triangulated with data from a qualitative study. The combined evidence suggests that when excess circulating aldosterone is reduced (adrenalectomy), or blocked (spironolactone), or aldosterone's salt and water retaining effects are minimised (amiloride), then nervous irritability and its subsequent psycho-behavioural manifestations are reduced. The effect however is slight and the conclusions are weakened by an apparent attrition bias, and the absence of a control group. Implications for further research are discussed.

Primary aldosteronism (PA) comprises about 11% patients referred to specialized centers for Arterial Hypertension. A considerable part of them carry a bilateral adrenocortical hyperplasia, also known as, idiopathic hypertension, which require life-long medical treatment. Notwithstanding this high prevalence rate the mechanisms leading to PA are unknown. However, anecdotal reports suggest that prorenin levels could be increased in PA, although the low or undetectable levels of active plasma renin, plasma prorenin levels could be increased. This suggests a possible role of prorenin in the pathophysiology of PA. The discovery of the prorenin receptor (PRR) in 2002, show a new way forward to the pathophysiology of the renin-angiotensin system: PRR can bind renin and prorenin inducing respectively an increase in renin catalytic activity and a non-proteolytically activation of prorenin. Till that discovery, prorenin was only seen as the inactive precursor of renin. Preprorenin is converted to prorenin in the juxtaglomerular cells of the kidney. The cleavage of the amino terminal 43-amino acid prosegment allows exposure of the active site in the secretory granules where generate renin. However the majority (75%) of prorenin is secreted constitutively; therefore The prorenin levels of human blood plasma are approximately 10-fold higher than those of renin. Further, they are increased in several conditions, including diabetes mellitus, where they could play a pathophysiologic role. PRR binding triggers activation of the mitogen activated protein (MAP) kinase–extracellular signal regulated kinase (ERK)1/2 and p38 signalling pathway independent from a possible generation of angiotensin II (Ang II). Activation of these signalling pathways, that are associated with cell proliferation and cell death has led to hypothesize that renin and prorenin could lead, via PRR, to hypertrophy and hyperplasia and ultimately to organ damage and cardiovascular events. In this study we first evaluated the gene and protein expression of PRR in adrenal gland and founded high expression of the PRR mRNA in APA, in human normal adrenal cortex and two human adrenocortical carcinoma cell lines: H295R and HAC15. Then, we sought for the expression of the PRR at the protein level with immunohystochemistry experiments on rat and human normal adrenal gland and with immunocytochemistry experiments. These experiments revealed a strong immunostaining for PRR in the adrenal gland at medulla and sub capsular level, and in aldosterone-secreting cells from the normal human ZG obtained by immunoseparation. In order to localize PRR we carried out immunoblot experiments on HAC15 and H295R cytosolic and membrane fractions and confocal microscopy experiments. These results showed that, PRR is mainly, but not exclusively, localized at the membrane level where partially colocalizes with the adhesion molecule CD56. We then investigated the subcellular localization of the PRR by immuno-gold electron microscopy experiments, and found that PRR is localized also in the nucleus, mitochondria and Golgi’s vesicles. We used H295R and HAC15 cell lines to investigate the functional relevance of this receptor. Stimulation with angiotensin II [100 nM], renin [50 nM] and prorenin [50 nM] induced ERK 1/2 phosphorylation. The renin and angiotensin II induced phosphorylation was abolished in presence of the angiotensin II (AT1) receptor antagonist irbesartan [5 μM] in both cell lines, whereas the phosphorylation induced by prorenin was abolished only in the H295R cells. These results suggest a role of PRR in proliferation, differentiation, and apoptosis of adrenal cortex and, therefore, a functional role of PRR in the pathophysiology of PA.
L’iperaldosteronismo primario (PA) colpisce l’11.2% dei pazienti ipertesi inviati ai centri dell’Ipertensione. Una quota considerevole di essi presenta un’iperplasia bilaterale del surrene, nota anche come iperaldosteronismo idiopatico, che richiede una terapia medica per tutta la vita. Nonostante l’alta prevalenza di questa patologia i meccanismi che ne stanno alla base sono tuttora sconosciuti. Tuttavia dati aneddotici suggeriscono che nel PA, a differenza dei livelli plasmatici di renina che sono ridotti o indosabili, i livelli di prorenina potrebbero essere aumentati. Ciò suggerisce la possibilità che la prorenina giochi un ruolo nella fisiopatologia del PA. Nel 2002 la scoperta del recettore della prorenina (PRR) ha aperto una nuova finestra sulla fisiopatologia del sistema renina-angiotensina: il PRR può legare sia la renina che la prorenina inducendo, rispettivamente, un aumento nell’attività catalitica della renina e un’attivazione non proteolitica della prorenina. Fino ad allora la prorenina era considerata solo il precursore inattivo della renina. La preprorenina viene convertita a prorenina nelle cellule juxtaglomerulari renali. Il clivaggio del prosegmento amino-terminale della prorenina permette l’esposizione del sito attivo nei granuli secretori generando renina. Tuttavia, la maggior parte (75%) della prorenina viene secreta costitutivamente; pertanto i livelli plasmatici di prorenina nell’uomo sono 10 volte maggiori di quelli della renina. Essi sono inoltre aumentati in diverse condizioni, tra cui il diabete mellito, dove potrebbero svolgere un ruolo fisiopatologico. Il legame di renina e prorenina al PRR attiva vie di signalling quali MAP (mitogen activated protein) chinasi-ERK 1/2 (extracellular signal regulated kinase) e p38 indipendentemente dalla generazione di angiotensina II (Ang II). Queste vie sono implicate nella proliferazione e apoptosi, il che ha fatto ipotizzare che renina e prorenina possano indurre ipertrofia e iperplasia e quindi danno d’organo ed eventi cardiovascolari via PRR. In questa ricerca abbiamo valutato l’espressione genica e proteica del PRR nel surrene e scoperto elevati livelli di espressione di mRNA del PRR negli APA, nella corteccia surrenalica umana normale e in due linee cellulari di carcinoma corticosurrenalico umano: H295R e HAC15. Abbiamo quindi investigato la presenza e localizzazione del PRR a livello proteico mediante immunoistochimica su surrene di ratto e umano e immunocitochimica. Ciò ha evidenziato un marcato immunostaining del PRR nel surrene, a livello midollare e sub capsulare, e in cellule di ZG umana secernenti aldosterone ottenute mediante immunoseparazione. Per localizzare il PRR, sono stati condotti esperimenti di immunoblot su frazioni di cytosol e membrana di cellule HAC15 e H295R ed esperimenti di microscopia confocale. Questi risultati hanno mostrato che il PRR è principalmente, ma non esclusivamente, localizzato a livello di membrana ove colocalizza parzialmente con la molecola di adesione CD56. Attraverso esperimenti di microscopia confocale immunogold abbiamo poi indagato la localizzazione subcellulare del PRR e scoperto che il PRR è localizzato anche nel nucleo, nei mitocondri e nelle vescicole del Golgi. Abbiamo quindi utilizzato H295R e HAC15 per studiare la rilevanza funzionale di questo recettore. La stimolazione con angiotensina II [100 nM], renina [50 nM] e prorenina [50 nM] ha indotto fosforilazione di ERK 1/2. In presenza dell’antagonista del recettore dell’angiotensina II (AT1) irbesartan [5 μM], la fosforilazione indotta da renina e angiotensina II era abolita in entrambe le linee cellulari mentre quella indotta dalla prorenina era abolita solo nelle H295R. Questi risultati suggeriscono un ruolo del PRR nella proliferazione, differenziamento e apoptosi del corticosurrene e quindi un possibile ruolo funzionale di questo recettore nella fisiopatologia dell’iperaldosteronismo primario.

Background. The detection of the mineralocorticoid receptor in parathyroid cells alongside recent evidences implicating the parathyroid hormone (PTH) in overproduction of aldosterone in primary aldosteronism in spite of suppression of the renin-angiotensin system indicate a relationship between parathyroids and the adrenocortical zona glomerulosa. The investigation of this relationship has been hampered due to lack of parathyroid cell line suitable for in vitro studies. Objective. To develop a technique to obtain a primary culture of human parathyroid cells with high yield and purity and to obtain subpopulations of chief and oxyphil cells. Results. After digestion of tissue from parathyroid adenomas, we isolated cells using an immunomagnetic bead method to remove fibroblasts and filtration devices to isolate cells by size. real-time qPCR evidenced expression of specific markers of parathyroid cells in cultured cells until day 10. At protein level, we found that some cells showed marked PTH immunostaining, whereas the remaining cells showed only a faint signal, suggesting the presence of two cell types. After loading cells with tetramethylrhodamine-methyl-ester (TMRM), we found two cell types, which differed for the fluorescent signal intensity. Conclusions. We found an optimal protocol to obtain a culture of human parathyroid cells that includes immunobinding-based technique and cellular filtration. Cultured cells retain their characteristic expression profile until day 10. Analysis of the mitochondrial uptake of TMRM and cell markers allows identification of two cell types forming the parathyroid tissue, i.e. oxyphil and chief cells
Nelle cellule di paratiroide è espresso il recettore mineralcorticoide Inoltre, nell’iperaldosteronismo primario i livelli sierici di ormone paratiroideo (PTH) sono aumentati. Questi dati unitamente al fatto che il PTH stimola la secrezione di aldosterone suggeriscono una relazione tra paratiroidi e zona glomerulosa del surrene. L'analisi di tale relazione è stata ostacolata finora dalla mancata disponibilità di una linea cellulare di paratiroide idonea per gli studi in vitro. Obiettivo. Investigare la posibilita di sviluppare una linea cellulare di paratiroide umana che permetta di investigare se, e attraverso quali meccanismi, la secrezione di PTH sia regolata dall’aldosterone. Risultati. Dopo digestione di tessuto ottenuto da adenomi di paratiroide sono state isolate le cellule utilizzando due metodi: 1) immunoseparazione mediante biglie incubate con CD90, anticorpo di membrana specifico per i fibroblasti, 2) separazione mediante filtrazione. L’espressione di marcatori propri delle cellule di paratiroide è stata confermata tramite real time qRT-PCR, microscopia elettronica e immunocitochimica nelle cellule in coltura fino al decimo giorno. Su la base del grado di immunostaining per il PTH e all'intensità di segnale dopo caricamento delle cellule con tetramethylrhodamine-metil-estere (TMRM), una sonda specifica per misurare il potenziale di membrana dei mitocondri.,due fenotipi cellulari sono stati identificati. Conclusioni. Con il protocollo messo a punto nel nostro laboratorio per isolare e coltivare cellule di paratiroide umana si è potuto accertare che le cellule della coltura primaria mantengono il loro fenotipo, caratterizzato dall'espressione dei marcatori propri delle cellule di paratiroide, fino al decimo giorno. Un processo di filtrazione, seguito dall'espressione dei marcatori cellulari e dall’analisi della captazione di TMRM mitocondriale consentono la separazione e l'identificazione delle due sottopopolazioni di cellule che formano il tessuto paratiroideo, cellule ossifile e cellule principali

Background and aim: hyperaldosteronism has been related to collagen deposition, myocardial fibrosis, and ventricular remodeling in experimental studies. More recent evidence suggest that these detrimental effects can develop independently of blood pressure and a significant decrease in the mortality rate of patients with heart failure who were treated with aldosterone antagonists has been reported. Primary aldosteronism (PA) is a form of secondary arterial hypertension that offers an important clinical opportunity for assessing the effects of hyperaldosteronism on the left ventricular (LV) anatomy and function because, in this condition, its effects are isolated from those of the renin-angiotensin axis. In the literature, only few longitudinal studies have evaluated cardiac changes after treatment of hyperaldosteronism with either surgical or medical treatment. Hence, in the present study we have explored the relationship between aldosterone and the heart by assessing cardiac anatomic and functional evolution of a large number of patients with PA surgically or medically-treated. Methods and Results: fifty-five patients with PA were enrolled in a prospective study and were followed for a mean of 6.4 years (range: 4.5 to 8 years) after adrenalectomy (n=41) or medical treatment (n=14). The diagnosis of APA (aldosterone producing adenoma) was based on adrenal vein sampling and pathology results and on follow-up data. At baseline and at follow-up we performed Doppler echocardiography for estimation of LV wall thickness and dimensions and transmitral LV filling flow velocity indexes. At baseline, PA patients who subsequently underwent adrenalectomy were younger and had lower body mass index and lower diastolic blood pressure than PA patients medically-treated. The former showed an excess LV hypertrophy and concentric remodeling, but with a less degree of diastolic dysfunction compared with the latter. At follow-up, despite a greater reduction of antihypertensive drugs in surgically treated patients (number of drugs: 1.7±1.4 vs 2.6±0.8 in surgically and medically-treated, respectively, p= 0.024) in both groups there was a significant reduction of blood pressure levels. A significant (p< 0.001) reduction in LV end-diastolic diameter, a reduction in LV mass index but with an increase in the relative wall thickness was observed in both groups. However, the last two parameters were significant only in the surgically treated PA. As regards the diastolic function, no significant modification was observed in the surgically treated PA. At variance, a significant (p= 0.002) reduction in the atrial contribution to LV filling was observed in medically-treated PA, possibly because they showed a higher degree of diastolic dysfunction at baseline. In both groups LV mass decrease was independent from pre-treatment blood pressure, body mass index, age, known duration of hypertension, follow-up interval and medical treatment. Conclusions: in PA patients, the excess aldosterone is associated with both increased LV wall thickness and mass. Treatment of hyperaldosteronism with either surgical or medical treatment induce a reduction in blood pressure levels, LV end-diastolic diameter, a reduction in LV mass index and an increase in the relative wall thickness, particularly in the surgically treated PA. With a similar fall of blood pressure and despite a greater reduction of antihypertensive drugs, these changes were more prominent in the adrenalectomized patients. Treatment can improve the diastolic function, but this was observed only in the medically-treated PA patients, likely because they had a higher degree of diastolic dysfunction at baseline.

L'hyperaldostéronisme primaire (HAP) est la plus fréquente cause identifiable de l'hypertension, et résulte de la production autonome d'aldostérone par les glandes surrénales. Chez la souris, la délétion génétique des canaux TASK1 et TASK3 provoque des changements biochimiques qui imitent HAP humain. Ces canaux permet la sortie de K+ et polarise le potentiel de la membrane des cellules glomérules. Nous avons étudié la variation et l'expression de KCNK3 et 9 chez l'homme. Notre étude d'association à montré aucune association d'HAP avec n'importe quel SNP au niveau de l'ensemble du génome. Le séquençage de l'ADN de la lignée germinale dans 825 cas d'HAP, et 41 échantillons d'ADN tumoral a abouti à 14 variantes différents dans KCNK3 et 9 dans la lignée germinale, dont 6 non-synonyme, 8 synonyme. Des tests in vitro n'ont montré aucune perte de la fonction du canal. Aucun changement de séquence somatique à été trouvé. L'hybridation-in-situ dans 6 glandes surrénales contrôle (CA) et 20 glandes adénomes produisant l'aldostérone (APA) a montré que KCNK3 été fortement exprimée dans les trois couches du cortex, tandis que l'expression de KCNK9 était faible et limitée au glomérule en CA. Dans les APA, l'expression de KCNK3 a été détectée, alors que l'expression KCNK9 était faible et hétérogène. Le transcriptome de 43 APA et 11 CA a révélé une légère surexpression de KCNK3 dans les APA, en corrélation avec l'expression de CYP11B2. La surexpression de TASK1 dans les APA peut être secondaire à un phénomène épigénétique. Alors que la variation de l'ADN est incompatible avec un rôle causal, il peut y avoir une possible contribution des changements d'expression de TASK1 dans HAP humain
Hypertension is the leading cause of human mortality globally. Representing about a tenth of all patients, Primary Aldosteronism (PA) is the commonest identifiable cause of hypertension, and results from the autonomous production of aldosterone by the adrenal glands. The two principal sub-types are Bilateral Adrenal Hyperplasia (BAH), and Aldosterone Producing Adenoma (APA), which account for two-thirds and one-third of the cases respectively. The molecular etiology of primary aldosteronism has remained elusive until recently, when through an exome sequencing study, mutations in the potassium channel-coding gene KCNJ5 were found to cause PA in humans. These mutations were found in up to 40% of APAs, and only in a rare familial variety of BAH. A subsequent exome sequencing study identified mutations in ATPase famile genes in about 7% of APAs, bringing the total genetic yield to about 47%. The molecular pathology of more than half of APAs and of most BAHs remain unexplained. In mouse models, the genetic deletion of TASK-1 and TASK-3 potassium channels cause biochemical changes that resemble those seen in human PA. TASK 1 and TASK 3 are background ‘leak’ potassium channels, which by permitting the outward flow of K+ ions, polarise the adrenal glomerulosa cell membrane potential. The genetic removal of these channels therefore results in a marked depolarization of the glomerulosa cells, leading to their increased aldosterone secretory function, diagnosed as PA. In humans, the contribution of TASK-1 and TASK-3 channel dysfunction to PA has been negated by sequencing studies of the genes that code for these channels (KCNK3 and KCNK9 respectively). However, these studies have included only a small number of patients, motivating a comprehensive molecular analysis of the genes in a large patient cohort. To this end, we investigated commonly and rarely occuring genetic variation in, and expression of, KCNK 3 and KCNK9. Our Genome Wide Association Study (GWAS) showed no association of PA (either APA or BAH subtypes or both) with any single SNP at the genome-wide level of statistical significance. At sub genome-wide levels, however, SNPs of KCNK3 did associate, and the association signal strengthened when specific combinations of the SNPs were tested for association at a time. While no inherited or acquired DNA sequence variation in KCNK3 and KCNK9 have ever been detected in PA patients, on sequencing germline DNA in 825 PA cases, and 41 tumoral DNA samples, 14 different coding single nucleotide variants in KCNK3 and KCNK9 were found in the germline DNA only, of which 6 were non-synonymous, and 8 synonymous. However, on heterologous expression and electrophysiology, these did not affect channel function. No somatic sequence changes were found.Expression of KCNK3 and KCNK9 was investigated by in-situ hybridization in 6 control adrenal glands and 20 adrenals from patients with APA. In the control adrenal, the KCNK3 gene was highly expressed in all three layers of the adrenal cortex, while KCNK9 expression was barely detectable, and restricted to the zona glomerulosa. In APAs, KCNK3 expression was detected in a majority of patients, while KCNK9 expression was low and heterogeneous among samples. Strikingly, KCNK9 was highly expressed in the hyperplastic peritumoral zona glomerulosa, possibly due to a positive feed-back by high circulating aldosterone or low potassium levels on KCNK9 expression. Transcriptome profiling of 43 APA and 11 control adrenals revealed a slight, but significantly increased expression of KCNK3 in adenomas compared to controls that correlated positively with CYP11B2 expression. The quantitative changes of TASK1 expression observed in APAs may be secondary to a primary epigenetic phenomenon or be secondary to increased aldosterone production due to dysregulation of master transcription factors or upstream signaling cascades in the aldosterone biosynthetic pathway

Background. Primary Aldosteronism (PA) is due to the presence of an Aldosterone-Producing Adenoma (APA) in almost two thirds of the cases. Due to the lack of a specific antibody for human aldosterone synthase (hCYP11B2), the functional identification of aldosterone-producing sites in the excised adrenal gland has not been possible thus far. To tackle this challenge we used novel monoclonal antibodies for hCYP11B2 and hCYP11B1 to immuno-phenotype a large series of adrenal glands excised from patients with PA, including APA that were diagnosed by the “four corners criteria” and genotyped for KCNJ5 mutations. Aims. Our aims were to identify the CYP11B2 and CYP11B1 steroidogenic patterns of the PA adrenal glands and to investigate whether immunostaining predicts the biochemical profile and/or the outcome of the patients. Design and Method. Double immunohistochemistry and immunofluorescence was used to detect CYP11B2 and CYP11B1 in the excised adrenals from PA patients. Quantification of CYP11B2 and CYP11B1 immuno-staining was performed using an observer-independent PC-assisted method specifically developed in our laboratory that takes into account the area fraction and the staining intensity of the markers. The staining intensity was reported as H-score value and evaluated for each potential aldosterone-producing structure, including CYP11B2 positive clusters, APA and nodules. Results. Based on the CYP11B2/CYP11B1 immunostaining of 111 excised adrenal glands from PA patients we could identify 5 major steroidogenic patterns (the proportion of cases shown in parentheses): Pattern 1: adenoma with uniform CYP11B2 staining and CYP11B1 staining outside the adenoma (16%); Pattern 2: adenoma with diffuse CYP11B2 staining, scattered CYP11B1 staining and some cells co-expressing CYP11B2/CYP11B1 inside the adenoma (46%); Pattern 3: adenoma expressing CYP11B1 and clusters of cells CYP11B2 positive (2%); Pattern 4: no adenoma, multiple clusters of sub-capsular CYP11B2 cells (25%); Pattern 5: multi-nodular adrenal cortex, clusters of sub-capsular CYP11B2 cells (11%). The rate of pattern 2 differed significantly between mutated and wild-type KCNJ5 tumors (p=.012) and gender (p=.005). CYP11B2 H-score in APAs reflects aldosterone production before surgery (r=0.388, p=.002); by contrast, CYP11B1 did not correlate with the cortisol production. After adrenalectomy, steroidogenic patterns and H-score did not predict different aldosterone values between the patients, but, pattern 3 was prevalently detected in patients who biochemically resolved the PA picture, but still present high blood pressure levels. Conclusions. Analysis of the excised adrenal gland from patients who were deemed to have an unilateral excess aldosterone production, by using specific antibodies for human CYP11B2 and CYP11B1, unveiled the existence of 5 different steroidogenic patterns. The H-score based on immunophenotyping which takes into account all aldosterone producing sites in the gland, including APA, clusters and nodules, reflected the aldosterone synthesis in the adrenal gland. Hence surgically-curable PA is far more heterogeneous than previously held, which challenges the classical notion that PA entails only APA or bilateral hyperplasia. CYP11B2 immuno-staining is a promising tool for gaining better understanding in the pathophysiology of PA disease.
Background. Nei centri di riferimento per l’ipertensione arteriosa più del 50% dei casi di iperaldosteronismo primario (PA) sono dovuti alla presenza di un Aldosterone-Producing Adenoma (APA). La surrenectomia determina la cura dall’ipertensione e la correzione del quadro biochimico di PA. La comprensione dei meccanismi che inducono lo sviluppo dell’APA è, pertanto, di fondamentale importanza clinica. La diagnosi di APA ad oggi è basata sul riconoscimento, all’interno della ghiandola surrenalica, di un nodulo costituito da cellule chiare, senza che si abbiano informazioni funzionali sulla produzione di aldosterone. La diagnosi di APA nel surrene rimosso rimane, quindi, piuttosto incerta. Lo sviluppo recente degli anticorpi monoclonali per l’aldosterone sintetasi (CYP11B2) e l’11beta-idrossilasi (CYP11B1) potrebbero permettere di localizzare le cellule responsabili dell’eccessiva produzione di aldosterone. Scopo. Lo scopo di questo studio è stato di identificare i patterns di espressione degli enzimi steroidogenici CYP11B2 e CYP11B1 nella ghiandola surrenalica da pazienti con PA e indagare se la caratterizzazione immuno-fenotipica possa predire il profilo biochimico e/o l’outcome pressorio di questi pazienti. Metodi. I surreni rimossi da 111 pazienti operati per PA che al cateterismo delle vene surrenaliche avevano mostrato eccesso di secrezione unilaterale, sono stati studiati mediante immunoistochimica e immunofluorescenza per CYP11B2 e CYP11B1. L’intensità della reazione cromogenica di CYP11B2 e CYP11B1 è stata quantificata utilizzando una metodica sviluppata nel nostro laboratorio che permette di attribuire ad ogni marker analizzato, un valore numerico denominato H-score, che è direttamente proporzionale alla percentuale di area positiva allo staining nella ghiandola e all’intensità di colorazione. Tutte le strutture potenzialmente competenti alla produzione di aldosterone (cluster di cellule CYP11B2 positive, APA e noduli) sono state analizzate per il calcolo dell’ H-score corrispondente. Risultati. L’analisi immunoistochimica nei pazienti PA ha permesso di identificare 5 patterns steroidogenici (la percentuale dei pazienti classificati nei patterns è espressa tra parentesi): pattern 1 comprendeva i surreni con un unico adenoma compatto e positivo per CYP11B2 e cellule positive per CYP11B1 nel tessuto adiacente all’adenoma (16%); nel pattern 2 sono stati classificati gli adenomi composti da cellule positive per CYP11B2 o CYP11B1 e alcune cellule che esprimevano contemporaneamente CYP11B2 e CYP11B1 (46%); i surreni classificati come pattern 3 presentavano un adenoma CYP11B1 positivo e alcuni cluster di cellule CYP11B2 positive (2%); nel pattern 4 non era presente un adenoma riconoscibile ma cluster di cellule CYP11B2 positive (25%); infine, i surreni del pattern 5 non mostravano alcun adenoma ma diversi noduli prevalentemente positivi per CYP11B1 e cluster di cellule CYP11B2 positive (11%). Al baseline, il pattern 2 si distingueva dagli altri patterns per la maggiore prevalenza di pazienti con una mutazione al KCNJ5 (p=.012) e di genere femminile (p=.005). L’H-score per CYP11B2 negli APA era direttamente proporzionale alla concentrazione di aldosterone (r=0.388, p=.002), mentre l’H-score per CYP11B1 non era correlato con la concentrazione plasmatica di cortisolo. Dopo la surrenectomia, né la classificazione in patterns né l’H-score sono stati in grado di predirre l’outcome biochimico dei pazienti. Tuttavia, i pazienti che dopo surrenectomia mostravano correzione del quadro biochimico ma continuavano ad essere ipertesi erano classificati prevalentemente come pattern 3. Conclusioni. Gli anticorpi specifici per CYP11B2 e CYP11B1 permettono di identificare cinque patterns immunofenotipici, molto variabili tra loro. La metodica sviluppata dal nostro gruppo per la quantificazione dell’H-score che tiene in considerazione tutte le strutture potenzialmente abili alla produzione dell’aldosterone, ovvero APA, clusters e noduli, fornisce informazioni accurate sulla produzione di aldosterone nei pazienti con PA. In conclusione, i risultati di questo studio mostrano che le forme di PA siano molto più eterogenee di quanto precedentemente ipotizzato, suggerendo che accanto all’APA classico definito come nodulo iper-funzionante e all’iperplasia esistano altri fenotipi che includono clusters e/o cellule che esprimono entrambi gli enzimi steroidogenici CYP11B2 e CYP11B1. L’immuno-staining dei surreni con hCYP11B2 sembra essere un promettente strumento non solo per la diagnostica ma anche per lo studio della fisiopatologia dell’iperaldosteronismo.

In a patient affected by primary aldosteronism we have identified the presence of a pheochromocytoma not secreting catecholamines. The analysis of the transcriptome in the pheochromocytoma revealed a high expression of Urotensin II (UII). UII is a somatostatin-like cyclic 11-aminoacid vasoconstrictor peptide, first identified in the teleost fish caudal-neuro-secretory system and then in humans, that has been demonstrated to be a potent vasoactive peptide involved in the physiology and pathophysiology of the cardiovascular system through mechanisms still largely unknown. Its action is now the subject of intensive studies on the effects it produces in different animal models and in different vascular beds. The direct effects on the myocardium such as ventricular hypertrophy, increased extracellular matrix and reduction of myocardial contractility suggest a role of UII in cardiac remodelling after myocardial infarction and heart failure. Even if the effects on cardiovascular system are still controversial UII has attracted considerable scientific interest aimed to investigate on its pathophysiology and on its the mechanism of action. We investigated among the expression of UII and its receptor in human adrenal glands and in different adrenal tumors. The opposite trend of expression of UII receptor obtained between aldosterone-producing adenoma and pheocromocitoma, along with the differences of genes implicated in UII signaling, supported a role of UII in the paracrine interactions between the adrenal medulla and cortex. Being relevant for the regulation of adrenal gland function and for the pathophysiology of pheocromocitoma and aldosterone-producing adenomas, these interactions might provide a mechanistic explanation for the pheocromocitomas that presented clinically as aldosteronism. We tested the “in vivo” effects of UII on blood pressure in rats, showing a delayed transient hypertensive effect that resembles the pressure trend in chronic infusion of aldosterone. Thus further supporting the hypothesis of a close interaction between UII and the renin-angiotensin-aldosterone system or even that UII acts through production of aldosterone as well as assess its hypertensive activity, its effect of cardiac hypertrophy and fibrosis and its role in regulating the growth of adrenocortical cells in rats. Considering that the mechanisms involved in the so called “escape phenomenon” seem responsible of the peculiar blood pressure trend in hyperaldosteronism we tested the blood pressure modifications in chronic infusion of UII counteracting the “escape phenomenon” through unilateral nephrectomy, high salt diet and concomitant infusion of spironolactone. The results obtained were promising: for the first time was shown an in-vivo not transient but continuous hypertensive effect of UII. The evidences obtained in blood pressure modification during chronic infusion of UII combined with the ones regarding UII and development of cortical tumors, has taken a step forward in identifying the mechanism of action of UII supporting the hypothesis of a possible close communication between medulla and adrenal cortex and a possible action of UII mediated by aldosterone. Additional investigations on the transgenic rat overexpressing UII in adrenal medulla will certainly contribute to verify our hypothesis.
In una paziente affetta da iperaldosteronismo primario abbiamo identificato la presenza di un feocromocitoma non secernente catecolamine. L’analisi del transcrittoma nel feocromocitoma ha rivelato un’elevata espressione di Urotensina II (UII). L’UII è un peptide ciclico di 11 aminoacidi, identificato inizialmente nel sistema neurosecretorio dei pesci teleostei e successivamente nell’uomo, che oggi è ritenuto un potentissimo vasocostrittore coinvolto nella fisiologia e nella fisiopatologia del sistema cardiovascolare i cui meccanismi di azione sono tuttora largamente sconosciuti. Molti studi su diversi modelli animali, sia in-vitro che in-vivo, hanno documentato che l’UII può indurre ipertrofia cardiaca, aumento della matrice extracellulare e riduzione della contrattilità miocardica, suggerendo una sua possibile implicazione nel rimodellamento cardiaco post-ischemico o nell’insufficienza cardiaca. Nonostante i suoi effetti sul cuore e sull’omeostasi pressoria siano tuttora controversi si è generato un notevole interesse nell’identificazione del ruolo fisiologico dell’UII e dei suoi meccanismi d’azione. Abbiamo studiato l’espressione dell’UII e del suo recettore nel contesto delle ghiandole surrenaliche e in diversi tumori surrenalici constatando un andamento pressochè opposto circa l’espressione di UII e del suo recettore tra feocromocitoma ed adenoma secernente aldosterone: nel feocromocitoma si è documentato un più alto contenuto di di UII rispetto all’adenoma secernente aldosterone che, viceversa, ha mostrato una maggiore espressione del recettore, suggerendo una down-regulation recettoriale secondaria alla produzione di UII. Queste evidenze suggeriscono un ruolo importante dell’UII nelle interazioni paracrine tra midollare e corticale surrenalica e nella fisiopatologia del pheocromocitoma e dell’adenoma secernente aldosterone, dando peraltro una spiegazione meccanicistica per quei feocromocitomi che si presentano clinicamente con iperaldosteronismo. Abbiamo inoltre testato in vivo l’effetto dell’UII sulla pressione arteriosa mediante la sua infusione cronica nel ratto documentando un andamento pressorio molto simile a quello che si ottiene nell’infusione cronica di aldosterone e caratterizzato da un iniziale effetto ipertensivo seguito da un adattamento pressorio verso i valori di partenza. Questo supporta ulteriormente l’ipotesi di stretta interazione tra UII e sistema renina-angiotensina-aldosterone o addirittura di un’azione dell’UII mediata dalla produzione di aldosterone. A questo proposito, partendo dal presupposto che il peculiare andamento pressorio negli stati di iperaldosteronismo è pesantemente influenzato dagli adattamenti di volume che si verificano grazie al fenomeno renale di “escape” abbiamo voluto verificare se vi fossero cambiamenti significativi nell’andamento pressorio durante l’infusione cronica di UII e la concomitante soppressione del meccanismo di escape. I risultati sono stati sorprendenti visto che per la prima volta si è documentato in vivo un effetto ipertensivo, non solo transitorio ma continuativo, dell’UII. Le evidenze ottenute, sia per quel che riguarda gli effetti dell’UII sulla pressione arteriosa che sullo sviluppo dei tumori surrenalici, sicuramente consentono di fare un passo in avanti circa l’identificazione del meccanismo d’azione a sostegno dell’ipotesi che l’UII possa agire come mediatore tra midollare e corticale surrenalica o mediante la produzione stessa di aldosterone. Gli studi in cui prevediamo di utilizzare ratti transgenici per l’espressione di UII nella midollare surrenalica potranno contribuire a confermare o confutare la nostra ipotesi.

Background: Primary aldosteronism (PA) is the most common endocrine form of secondary arterial hypertension with an estimated prevalence of ~ 11.2% in patients referred to specialized centers and 4% in primary care, but as high as 50% in patients with resistant hypertension. The two main forms of PA are: aldosterone-producing adenoma (APA), surgically curable with the removal of adenoma that is able to overproduce aldosterone, and bilateral adrenocortical hyperplasia (BAH), which needs drug therapy. About ~ 30% APAs have somatic mutations in KCNJ5 gene that encodes for the potassium channel (KIR3.4), which plays a crucial role in the maintenance of the cell membrane by pumping potassium (K+) out of the cells, thereby producing a negative membrane potential. The KCNJ5 channels that contain G151R, T158A, or L168R mutations cause membrane permeability to Na+, resulting in Na+ entry, cell depolarization, constitutive aldosterone production, and cell proliferation. The standard approach used to detect the mutations in KCNJ5 gene is sequencing of DNA extracted from adrenal tissue of hypertensive patients with lateralized excess in aldosterone production. However, DNA sequencing is a time consuming and rather expensive technique not feasible in all standard laboratories. Aims of our study were 1) to develop a strategy for genotyping DNA extracted from the adrenal tissue, which exploited a TAQ-MAN based PCR technology; 2) to evaluate if such Taq-Man-base technology allows detection of KCNJ5 mutations in the adrenal tissue and cf-DNA isolated from peripheral blood. Methodologic Approach: 1. Development of a novel strategy based on Taq-man probe to detect KCNJ5 mutations 2. Development of a protocol to isolate cf-DNA from blood collected in the inferior vena cava and adrenal veins. 3. Measurement of cf-DNA concentration and assessment of its fragmentation. 4. Identification of KCNJ5 mutation in cf-DNA Results: By applying the novel technology based on Taq-man probe we correctly identified 30 mutated patients in a cohort of 50 consecutive APA patients, with no misclassification. After isolating cf-DNA from the adrenal veins and inferior cava blood, and measuring its concentration, we evaluated cf-DNA fragmentation with the integrity index in 24 samples. Integrity index < 1 suggested that cf-DNA was released from the adrenal tissue through an apoptotic mechanism. HRM analysis of cf-DNA isolated from adrenal blood allowed us to identify the KCNJ5 mutation in the APA side. Conclusions and perspectives: The novel technology based on Taq-man probe allowed detection of all mutated patients in the examined cohort of APA patients, thus proving that this strategy could be used as an alternative to DNA sequencing. The results of our study also showed the feasibility to isolate cf-DNA from small amount of blood collected from the adrenal veins, and suggested that KCNJ5 mutations may be detected in cf-DNA. However, this approach needs to be verified for a larger population to determine feasibility and accuracy. If confirmed, analysis of cf-DNA isolated from the peripheral venous blood could be helpful for an early detection of KCNJ5 mutations and therefore for the selection of PA patients to be submitted to adrenal vein sampling. Furthermore, this strategy could be also useful for detecting KCNJ5 germline mutations responsible for the rare hereditary form of hyperaldosteronism FH-3.
Backgouund. L’Iperaldosteronismo primario (PA) è la forma endocrina più comune d’ipertensione arteriosa secondaria con una prevalenza stimata di circa il 4% nella popolazione generale e dell’11% nei pazienti che afferiscono ai centri di riferimento per l’ipertensione. Nei pazienti con ipertensione resistente la prevalenza del PA è stimata pari al 50%, mostrando che tale patologia non è così rara come ritenuto in passato. Le due forme principali di PA sono l’adenoma producente aldosterone (APA), caratterizzato da iperproduzione lateralizzata di aldosterone, e l’iperplasia surrenalica bilaterale (BAH). La distinzione tra le due forme è di cruciale importanza poiché la prima richiede terapia chirurgica, mentre la seconda terapia medica. Considerando che la rimozione dell’APA determina la correzione del quadro biochimico-clinico di PA e la cura o il miglioramento dell’ipertensione, il riconoscimento dell’APA è fondamentale per offrire una chance di guarigione dell’ipertensione o di miglioramento del controllo dei valori pressori ai pazienti che ne sono affetti. Il 40% degli APA presenta mutazioni somatiche nel gene KCNJ5 che codifica per il canale del potassio KIR 3.4. Questo canale gioca un ruolo fondamentale nel mantenimento del potenziale di membrana pompando il K+ al di fuori della cellula, provocando in tal modo un potenziale di membrana negativo. Allorquando il gene KCNJ5 contiene le mutazioni G151R, T158A o L168R il canale KIR 3.4 acquisisce capacità di condurre Na+ all’interno della cellula. Gli effetti della mutazione a livello della cellula sono depolarizzazione cronica, produzione costitutiva di aldosterone e proliferazione cellulare. L’approccio attualmente in uso per identificare tali mutazioni è il sequenziamento, secondo Sanger, del DNA estratto dal tessuto surrenalico rimosso durante surrenectomia nei pazienti con PA e iperproduzione lateralizzata di aldosterone. Il sequenziamento del DNA, tuttavia, è costosa richiede tempo e non è disponibile di routine in tutti i laboratori. Scopo generale dello studio è stato quello di sviluppare una strategia alternativa al sequenziamento che preveda l’uso delle sonde Taq-man in Real Time PCR (Q-PCR) per il rilevamento di mutazioni KCNJ5 nel tessuto surrenalico e nel DNA circolante (cell-free DNA, cf-DNA) isolato da sangue periferico. Lo sviluppo di questa metodologia potrebbe semplificare notevolmente l’identificazione delle mutazioni KCNJ5 negli APA e, infine, permetterne la detenzione nel DNA del sangue circolante. In sintesi, l’approccio metodologico include: sviluppo di una nuova strategia basata sull’utilizzo delle sonde Taq-man per rilevare le mutazioni nel gene KCNJ5. Sviluppo di un protocollo per isolare il cf-DNA da sangue delle vene surrenaliche e dalla vena cava inferiore. Misurazione della concentrazione del cf-DNA valutandone la sua frammentazione. Identificazione di mutazioni nel gene KCNJ5 a partire dal cf-DNA Risultati. Applicando la tecnologia sviluppata nel nostro laboratorio, basata sulle sonde Taq-man, sono stati identificati correttamente 30 pazienti mutati in una coorte di 50 pazienti APA consecutivi, senza errori di classificazione. Dopo aver isolato il cf-DNA dal sangue delle vene surrenaliche e dalla vena cava inferiore, e misurato la sua concentrazione, abbiamo valutato la frammentazione del cf-DNA in 24 campioni con l'indice di integrità. I bassi valori dell’indice d’integrità riscontrati nei cf-DNA isolati da sangue venoso surrenalico suggeriscono che la ghiandola surrenalica rilasci per apoptosi frammenti di DNA. L’analisi HRM dei cf-DNA isolati dal sangue delle vene surrenaliche di un paziente con un APA sinistro contenente la mutazione L168R ha permesso d’identificare correttamente la mutazione nel cf-DNA isolato dalla vena surrenalica sinistra. Conclusioni e prospettive. La tecnologia basata sulle sonde Taq-man ha permesso d’identificare, senza errori di misclassificazione, in una coorte di 50 pazienti con APA, tutti i 30 pazienti che presentano una mutazione del gene KCNJ5. Tale strategia, pertanto, potrebbe rappresentare un’alternativa alla ben più lunga e complessa tecnica basata sul sequenziamento del DNA. I risultati del nostro studio hanno anche mostrato che è possibile isolare il cf-DNA da esigue quantità di sangue raccolto dalle vene surrenaliche permettendo l’identificazione delle mutazioni KCNJ5 usando il cf-DNA tramite approccio combinato sonde Taq-man e analisi HRM. Quest’ultimo approccio, che prevede l’uso del cf-DNA richiede, tuttavia, conferma in un ampio numero di soggetti. La stessa strategia potrebbe anche essere impiegata in futuro per la rilevazione di mutazioni germinali KCNJ5 responsabili della nota forma ereditaria d’iperaldosteronismo FH-3.

Primary aldosteronism (PA), a common form of secondary hypertension, is characterized by an excess autonomous aldosterone secretion. In a percentage ranging from a half to two thirds of the cases it is due to a surgically curable aldosterone-producing adenoma (APA) and in the rest to bilateral adrenal hyperplasia. The molecular mechanisms underlying aldosterone hypersecretion are unknown. Recent evidences suggest that amino acid residue substitutions in the selectivity filter of the Kir3.4 (KCNJ5) potassium channel may cause a constitutive aldosterone secretion from aldosterone-producing adenomas (APA). Such somatic mutations were also found to be associated with higher plasma aldosterone concentrations in the patients with an APA, thereby suggesting a causative role of the mutations in the development of APA and hyperaldosteronism. Hence, we performed a study with the aims to search for KCNJ5 mutations in APA patients referred to two Italian referral centers. Through this search we could also identify a novel KCNJ5-insT149 mutation out of the selectivity filter that was a fully characterized from the electrophysiological and phenotypic standpoint. APA samples (n=195) from consecutive patients with a conclusive diagnosis of APA were screened by high melting resolution curve for KCNJ5 mutations. We found that the mutations occurred in 24.6% of patients. These findings were confirmed by Sanger sequencing. The mRNA content of CYP11B2, but not of CYP11B1, and plasma aldosterone and, accordingly, the lateralization index were higher (P < 0.02) in the APA with the mutation than in the APA without such mutations. A novel c.446insAAC insertion resulting in the mutant protein KCNJ5-insT149 was identified In a patient presenting with severe drug-resistant hypertension. To functionally characterize this novel KCNJ5 channel mutation a mutated cDNA harbouring c.446insAAC insertion was generated by site-directed mutagenesis and transfected in mammalian cells. KCNJ3 cDNA was also transfected into the same cells to reproduce the tetrameric structure of the KCNJ3/KCNJ5 channel. CYP11B1, CYP11B2 and 17α-hydroxylase were localized in the adrenal gland of the mutated APA patient with immunohistochemistry and immunofluorescence. CYP11B2 mRNA levels and aldosterone concentrations were also measured to investigate the impact of the mutation on the secreting activity. By using a whole-cell patch clamp technique and molecular modeling we explored membrane Na+ and Ca2+ currents and created a 3D image of the insT149 KCNJ5 channel. Compared to wild type and mock-transfected HAC15 adrenocortical cells, those expressing the mutant KCNJ5 showed increased CYP11B2 expression and aldosterone secretion. Likewise HEK293 expressing the mutated KCNJ5-insT149 channel exhibited a 2-fold increase in intracellular Na+ and a substantial rise in intracellular Ca2+ caused by activation of voltage-gated Ca2+ channels. Hence, the novel KCNJ5 K+ channel mutation induces abnormal Na+ permeability, membrane depolarization, a rise in cytosolic Ca2+ and increased aldosterone synthesis. Thus, our findings support the concept that channelopathies involving the KCNJ5 K+ channel mechanistically account for constitutive secretion of aldosterone in human APA.
L’ iperaldosteronismo primario (PA) è la causa più frequente di ipertensione secondaria ed è caratterizzato da una secrezione elevata ed autonoma di aldosterone. Le due forme principali sono l’iperplasia surrenalica bilaterale e l’adenoma secernente aldosterone. I meccanismi molecolari alla base dell’ipersecrezione di aldosterone sono tuttora sconosciuti. Tuttavia recenti studi hanno dimostrato che sostituzioni amminoacidiche all’interno del filtro di selettività del canale del potassio Kir3.4 (KCNJ5 possono provocare una secrezione autonoma di aldosterone in adenomi producenti aldosterone (APA). Tali mutazioni somatiche sono associate ad alti livelli plasmatici di aldosterone nei pazienti con APA, suggerendo un ruolo causale di tali mutazioni nello sviluppo di APA e iperaldosteronismo. Pertanto abbiamo condotto uno studio in pazienti affetti da APA afferenti a due centri di riferimento italiani, effettuando lo screening per le mutazioni somatiche di KCNJ5, ed abbiamo individuato e caratterizzato la mutazione KCNJ5-insT149, mai descritta in precedenza. Mediante analisi ad alta risoluzione delle curve di melting per le mutazioni in KCNJ5 sono stati studiati 195 pazienti consecutive con una diagnosi conclusiva di APA. Il 24,6% dei pazienti presentava una mutazione nel filtro di selettività del KCNJ5, tale prevalenza è stata confermata mediante sequenziamento Sanger. Nei pazienti affetti da mutazione di KCNJ5 l’espressione genica di CYP11B2 (29,9 ± 7,4 vs 10,3 ± 3,6, P <0,02), ma non quella di CYP11B1, risultava superiore rispetto ai pazienti non affetti da mutazioni, lo stesso valeva per l’indice di lateralizzazione. In un paziente con ipertensione farmaco-resistente grave è stata identificata l’ inserzione c.446insAAC, che codifica per la proteina mutante KCNJ5-insT149. Per caratterizzare funzionalmente questa nuova mutazione, attaverso mutagenesi sito-diretta, è stato generato un cDNA codificante per il canale KCNJ5 mutato e trasfettato in cellule di mammifero. Il cDNA codificante KCNJ3 è stato transfettato insieme a quello per KCNJ5 in modo da riprodurre la struttura tetramerica del canale KCNJ3/KCNJ5. CYP11B1, CYP11B2 e 17α-idrossilasi sono stati rilevati attraverso tecniche di immunoistochimica e immunofluorescenza nella ghiandola surrenale del paziente. L’espressione genica di CYP11B2 e le concentrazioni di aldosterone sono stati misurati per studiare l'impatto della mutazione sull'attività secernente. Utilizzando la tecnica di “whole-cell patch clamp e modeling molecolare” abbiamo studiato le correnti trans-membrana di Na+ e Ca2+ e generato una immagine 3D del canale insT149 KCNJ5. Rispetto al wild type e alle cellule adrenocorticali HAC15, le cellule transfettate con KCNJ5-insT149 esprimevano alti livelli del gene CYP11B2 e mostravano un’aumentata produzione di aldosterone. Allo stesso modo cellule HEK293 che esprimono il canale KCNJ5-insT149 mutato mostravano un aumento pari a due volte di Na+ intracellulare e un aumento sostanziale di Ca2+ intracellulare in seguito all’ attivazione dei canali del Ca2+ voltaggio-dipendenti. Quindi, la nuova mutazione del canale del K+ KCNJ5 induce un’anomala permeabilità della membrana al Na+, depolarizzazione della membrana, un aumento di Ca2+ intracellulare e aumento della sintesi di aldosterone. I nostri risultati nel complesso supportano il concetto che le canalopatie che coinvolgono il canale del K+ KCNJ5 sono alla base della secrezione costitutiva di aldosterone in pazienti affetti da APA.

Primary aldosteronism is the most common secondary cause of hypertension with a prevalence of about 10%. About half of PA cases are caused by aldosterone-producing adenomas (APA). Two APA subtypes, ZG-like and ZF-like APAs, have been described, according to the histological resemblance to normal zona glomerulosa (ZG) and zona fasciculata (ZF), underlying somatic mutations (KCNJ5 commonly found in ZF-like, CACN1AD, ATP1A1, ATP2B3, CTNNB1 in ZG-like APAs), and transcriptome profile. It is unknown if the process of tumorigenesis differs between ZG- and ZF-like APAs. In order to define ZG specific genes, we have compared the transcriptome of APAs and their adjacent adrenal glands by microarray assay. RNA was isolated by laser capture microdissection (LCM) from adjacent ZG, ZF and APAs from 14 patients with Conn’s and 7 patients with phaeocromocytoma. Two top hit genes from the comparison of ZG vs ZF were functionally studied, ANO4 and NEFM. NEFM, encoding neurofilament medium, was the fourth most up-regulated gene in ZG vs ZF, showing 14.8-fold-fold higher expression levels (p=9.16-12) in ZG than ZF. NEFM was also one of the most down-regulated genes in ZF-like vs ZG-like APAs. Immunohistochemistry (IHC) confirmed selective high expression of NEFM in ZG and ZG-like APAs. Silencing NEFM in H295R cells increased aldosterone secretion and cell proliferation. In addition, it increased stimulation and inhibition, respectively, of aldosterone secretion from H295R cells by the dopamine receptor D1R agonist fenoldopam and antagonist SCH23390. IHC showed predominantly intracellular staining for D1R in NEFM-rich ZG-like APAs, but membranous staining in NEFM-poor ZF-like APAs. Aldosterone secretion in response to fenoldopam in primary cells from ZG-like APAs was lower than in cells from ZF-like APAs. NEFM expression levels directly correlate with KCNJ5 phenotype: KCNJ5 mutations down-regulate NEFM mRNA and protein levels in H295R cells and in primary cells from ZG-like APAs. ANO4,encoding a Ca2+-activated chloride channel family member, was the third most upregulated gene, showing 19.9-fold higher expression levels (p=6.6x10-24) in ZG than ZF. IHC confirmed ZG selectivity of ANO4 protein in the adrenal cortex. The staining was mainly cytoplasmic. Unlike NEFM, there was no difference in expression of ANO4 between ZG- and ZF-like APAs, the levels being mid-way between those of ZF and ZG. Overexpression of ANO4 in H295R cells caused an increase in CYP11B2 and NR4A2 gene expression levels but basal aldosterone secretion was unchanged. In the presence of calcium agonists, ANO4 reduced aldosterone secretion. ANO4 subcellular localisation was confirmed as cytoplasmic by immunofluorescence microscopy of transfected cells. When exposed to calcium ionophores, ANO4 generated small chloride currents as detected by YFP assay. In summary, the comparison of transcriptome of ZG with paired ZF found unexpected up-regulated genes. Most of the highly up regulated genes in human ZG, including NEFM and ANO4, inhibit either basal or stimulated aldosterone secretion, and this may reflect an adaptive response to high salt intake. No clear-cut correspondence was found between transcriptome of APAs and their resembling zone of adrenal cortex. The down-regulation of NEFM following transfection of mutant KCNJ5 suggests that ZF-like properties may be a consequence of mutation, rather than tissue of origin.

In patients with primary aldosteronism (PA), adrenal vein sampling (AVS) is considered the gold standard to distinguish between unilateral and bilateral autonomous production of aldosterone, while diagnostic imaging tests by CT scan or MRI are useful but often inconclusive for the diagnosis of PA. To date agreement is lacking on the best criteria indicating successful cannulation and lateralization. The aim of the study was to evaluate the impact of different diagnostic criteria for the successful cannulation and lateralization on subtype diagnosis and to compare the difference of the findings between adrenal CT scan and AVS. Sixty-six patients with confirmed PA underwent AVS. The different diagnosis of PA subtype reached using AVS data assessed by more permissive (type 1) and strict (type 2) criteria were compared. Al patients performed CT scan before AVS and imaging results were compared with results of AVSs (using both criteria). Using Type 1 criteria AVSs were successful in 86,3% of patients, while they were successful in only 66,6% using type 2 criteria. Type 1 criteria also led to a higher rate of diagnosis of unilateral PA (80,7% of successful procedures) than type 2 (75% of successful procedures). There was considerable disparity in the diagnosis reached using the 2 different criteria, with a concordance in only 50% of patients. In conclusion more permissive criteria for successful cannulation and lateralization on AVS can lead to incorrect diagnosis and accordingly to inappropriate treatment options. In the selected group of patients with successful AVS, CT findings correlated with AVSs findings and correctly identified unilateral or bilateral disease in 57,9% of patients using type 1 criteria and in 47,7% using type 2 criteria. Finale diagnosis was based on histological results in 29 patients (43,9 %) which underwent adrenalectomy based on AVSs findings. On the basis of CT findings alone 17,5% of patients from the first group and 31,8% of patients of the second group probably would have been incorrectly bypassed as candidates for adrenalectomy. CT scanning lacks sensitivity and specificity and should, therefore, be followed by AVS, which is the only reliable means of differentiating unilateral from bilateral PA and lateralizing APAs preoperatively. However, there are still controversies to be solved by large prospective studies on the criteria to adopt for defining the most appropriate cut off for both correct cannulation and lateralization.
Il Cateterismo venoso surrenalico (AVS) è considerato ad oggi il gold standard per distinguere tra una ipersecrezione aldosteronica unilaterale o bilaterale in pazienti affetti da iperaldosteronismo primitivo (PA); mentre procedure di imaging, quali TAC o RMN pur essendo molto utili spesso risultano inconclusive per la diagnosi. Tuttavia manca ancora un accordo su quale criterio diagnostico usare per identificare un corretto incanalamento ed una corretta lateralizzazione al AVS. Scopo principale del presente studio è di valutare l'impatto di differenti criteri utilizzati per definire il successo di incanulazione e la lateralizzazione nella diagnosi di sottotipo e di analizzare il ruolo del cateterismo delle vene surrenaliche nella diagnosi e nella conseguente scelta di trattamento di iperaldosteronismo primitivo, comparando risultati di imaging ottenuti tramite TAC surrenalica con l'esito dei cateterismi venosi surrenalici. 66 pazienti con diagnosi di PA sono stati sottoposti ad AVS. I risultati ottenuti sono stati interpretati usando due criteri per l'identificazione di incanalamento e di lateralizzazione diversi, uno più permissive (Tipo 1) e l'altro più restrittivo (Tipo 2). Tutti I pazienti hanno eseguito una TAC surrenalica prima del AVS e le immagini sono state confrontate con I risultati ottenuti al cateterismo (usando entrambi criteri). Usando il criterio tipo 1 il cateterismo è stato eseguito con successo nel 86,3% dei pazienti, mentre ha avuto successo solamente nel 66,6% dei casi usando il criterio di interpretazione tipo 2. Il criterio tipo 1 ha portato ad identificare un numero maggiore di APA (80,7% delle procedure eseguite con successo) rispetto al criterio tipo 2 (75% delle procedure eseguite con successo). Una diagnosi concorde usando I due criteri si è ottenuta solamente nel 50% dei pazienti. In conclusione criteri più permissive per valutare il successo di incanalamento e la lateralizzazione al AVS possono portare a diagnosi incorrette e conseguentemente a scelte terapeutiche inappropriate. Nel gruppo di pazienti in cui il cateterismo è stato eseguito con successo, la TAC correlava con I risultati del AVS identificando correttamente lesione uni-o bilaterali nel 57,9% dei casi usando il criterio di tipo 1, e nel 47,7% dei casi usando il criterio tipo 2. La diagnosi finale è basata sull’analisi isto-patologica in 29 pazienti ( 43,9%) sottoposti a surrenectomia monolaterale. Sulla base della sola immagine TAC il 17,5% dei pazienti del primo gruppo e il 31,8% del secondo gruppo sarebbe stato sottoposto erroneamente a surrenectomia. La TAC presenta minore sensibilità  e specificità  e dovrebbe precedere un AVS, che rappresenta il metodo più idoneo nell'identificare un PA unilaterale o bilaterale. Tuttavia permangono tuttora delle controversie che dovrebbero essere risolte da studi prospettici a largo spettro per definire I cut-off più appropriate per identificare un corretto incanalamento e corretta lateralizzazione.

L’hyperaldostéronisme primaire [HAP] résulte d’une hypersécrétion d’aldostérone d’origine surrénale. La compréhension de la pathogénie de cette maladie, dont la prévalence est estimée à 10% de la population hypertendue, est essentielle pour le développement de nouveaux outils diagnostiques et thérapeutiques. Dans ce contexte, ce travail de doctorat avait pour but d’identifier de nouvelles orientations thérapeutiques en testant un inhibiteur de l’aldostérone synthase et de rechercher de nouveaux marqueurs diagnostiques par l’étude du profil d’expression des microARN [miRs]. Dans une étude de phase II, 14 patients présentant un HAP ont reçu un inhibiteur de l’aldostérone synthase : le LCI699 pendant 4 semaines. Nous avons ainsi pu montrer que le LCI699 permet de diminuer les concentrations d’aldostérone de 70 à 80% et de normaliser la kaliémie chez tous les patients. En revanche, il n’a qu’un effet modéré sur la pression artérielle et sur l’élévation des concentrations de rénine, et n’est que partiellement sélectif pour l’aldostérone synthase. De plus son efficacité est moindre que celle de l’éplérénone, antagoniste minéralocorticoide administré aux mêmes patients au décours du LCI699. Nous avons ensuite étudié l’expression de 754 miRs dans des adénomes produisant de l’aldostérone [APA] et dans des surrénales contrôles. L’hypothèse était qu’une dérégulation de leur expression pouvait être impliquée dans la tumorigénèse et la surproduction d’aldostérone. L’objectif secondaire était d’identifier des miRs utilisables en tant que biomarqueurs. Cette analyse par carte microfluidique a révélé que 27 miRs sont significativement sous exprimés dans les APA et un seul miR est surexprimé. L’expression différentielle de deux de ces miRs : miR 137 et miR 375 a pu être confirmée dans une cohorte de validation de 36 APA: Des résultats préliminaires in vitro indiquent que le miR 375 pourrait induire une diminution de la synthèse d’aldostérone. Enfin, l’analyse de l’expression de ces miRs dans le plasma a permis de mettre en évidence une sous-expression du miR 375 chez les patients atteints d’HAP en comparaison à des sujets sains. En conclusion, le blocage de la biosynthèse de l’aldostérone représente une nouvelle option thérapeutiques, cependant il est nécessaire de développer une seconde génération de molécules : plus puissantes et plus sélectives. Les analyses effectuées sur les APA ouvrent de nouvelles perspectives pour l’identification de nouveaux biomarqueurs tels que les miRs circulants
Primary aldosteronism [PA] results from the hypersecretion of aldosterone by the adrenals. Understanding the pathogenesis of the disease is essential for identifying new diagnostic and therapeutic tools. In this context the purpose of my PHD was to investigate the effects of an aldosterone synthase inhibitor and second to investigate new diagnostic options by the extensive study of microRNA [miRNA]. In a phase II clinical study, 14 patients with PA were administered an aldosterone synthase inhibitor: LCI699. Four weeks of treatment lead to a 70 to 80% decrease in aldosterone concentration, associated with the cure of hypokalemia. However, there was only a mild effect on blood pressure and volemia (reflected by renin concentration). In addition, these results demonstrated an incomplete selectivity of LCI699 for aldosterone synthase in vivo, and showed that LCI699 is less potent than the blocker of the mineralocorticoid receptor: eplerenone . We also characterized the miRNA profile of Aldosterone producing adenomas [APA]. The hypothesis was that a dysregulation of the expression of miRNA could induce tumorigenesis and increase the production of aldosterone. The secondary aim of the study was to identify miRNA that could be measured in plasma as biomarkers. miRNA profiling of 754 miRNA using quantitative PCR Low Density array, revealed 28 miRNA whose expression was significantly different in APA. The differential expression of two miRNA: miRNA 137 and miRNA 375 was confirmed in a validation cohort of 36 APA. Preliminary in vitro studies showed that up-regulation of intracellular levels of miR 375 may reduce aldosterone secretion in H295R cells. Lastly, circulating plasma levels of miR 375 are differentially expressed between patients with PA and healthy volunteers. In conclusion, the blocking of the aldosterone pathway in hypertensive patients is a novel therapeutic option but second-generation drugs more potent and more selective of aldosterone synthase are required. Profiling miRNA in APA offers new prospect for the development of biomarkers, such as measuring circulating miRNA in plasma

博士
國立臺灣大學
臨床醫學研究所
100
Primary aldosteronism (PA) is a curable and common hypertensive disease, and is characterized by an inappropriate production of aldosterone. PA, the prevalence of which is around 5-10% of patients with hypertension, consists of two subtypes: aldosterone-producing adenoma (APA) and idiopathic hyperaldosteronism (IHA). Patients with PA have been shown to have a higher prevalence of cardiovascular events and atrial fibrillation than patients with essential hypertension (EH), which are independent of blood pressure effects. In addition, patients with PA have a more prominent left ventricular (LV) mass, and a higher degree of LV diastolic function impairment and myocardial fibrosis than those with EH. In recent studies, macrophage-mediated low grade inflammation has been shown to play an important role in aldosterone-inducing cardiac fibrosis. However, the mediators of this process are still unclear. In order to investigate the change of cardiac structure and texture in PA patients and their relation to macrophage, we designed a series of studies. In the part, we found that serum potassium level was negatively associated with LV mass index (LVMI) in PA patients. Compared with patients with EH, PA patients had a greater impairment of cardiac diastolic function, which was independent of serum potassium level. In the second part, we found that LV hypertrophy in PA patients could be significantly reversed by adrenalectomy. Pre-operative LVMI and change of systolic blood pressure were associated with the degree of LVMI decrease. In the third and fourth part, we found that adrenalectomy reversed not only LV geometry but also altered myocardial texture in patients with unilateral hyperaldosteronism. This suggests that increases in collagen content in the myocardium of patients with unilateral hyperaldosteronism might be reversed by adrenalectomy. In the fifth part, we investigated whether aldosterone induced macrophage to secret galectin-3 and its mechanisms. We found that aldosterone induced the THP-1 cell to secret galectin-3 via mineralocorticoid receptors. The induced secretion occurred through the PI3K/Akt pathway, and NF-kB was an important transcriptional factor in this pathway. In the sixth part, we investigated whether aldosterone induced macrophage to secret IL-6 and its mechanisms. We found that aldosterone induced the THP-1 cell to secret IL-6 via mineralocorticoid receptors. The induced secretion was achieved through the PI3K/Akt and p38 pathway, and NF-kB and NF-IL-6 were important transcriptional factors in this pathway. In the seventh part, we found that patients with APA had higher plasma IL-6 levels than EH patients’ and the increased plasma IL-6 concentration decreased significantly one year after adrenalectomy in the APA patients. However, plasma galectin-3 levels were comparable in both groups, and there was also no change one year after adrenalectomy in the APA patients. As hypertension is an important risk factor of cardiovascular disease, and as PA is a curable and common hypertensive disease, the issues of PA are becoming increasingly important. It is hoped that these studies will promote a greater understanding of PA and its effect on the cardiovascular system. Key words: primary aldosteronism; aldosterone; left ventricular hypertrophy; cardiac fibrosis; IL-6; galectin-3

碩士
國立臺灣大學
臨床醫學研究所
103
Primary aldosteronism (PA) was one of the most common causes of secondary hypertension. Exposure to excessive aldosterone led to increased arterial stiffness. The effect of adrenalectomy on arterial stiffness reversibility was seldom reported. Besides, the time course of arterial stiffness reversal and the actual predicting factors on arterial stiffness reversal were still unclear. We prospectively enrolled 102 patients with aldosterone producing adenoma (APA) from March 2006 to January 2012. We measured the pulse wave velocity (PWV) between brachial-ankle (baPWV) and heart-ankle (haPWV) before, and 6 months and 12 months after adrenalectomy of all enrolled patients. All other physiological and biochemistry parameters were collected at the same time and analyzed after the 1-year follow-up. The baseline baPWV averaged as 1636± 310 cm/s while haPWV as 1096±138cm/s. The determinant factors to baseline baPWV were age, duration of hypertension, and baseline systolic blood pressure (SBP) after multi-variable linear regression analysis. Similarly, the determinant factors for baseline haPWV were age, duration of hypertension, baseline systolic pressure and diastolic pressure (DBP). After treatment, the patients’ PWV decreased significantly during the first 6-month (baPWV 1533 ± 278 at 6-month, p < 0.001; haPWV 1048±129 at 6-month, p<0.001). No further reduction was noted during the following 6 month (baPWV 1562±31 at 12-month, p = 0.248; haPWV 1049±130 at 12-month, p=0.415). After multi-variable regression analysis, the determinants of baPWV decrement at 6 months (ΔbaPWV0-6mo) were decrement of diastolic blood pressure at 6 months (ΔDBP0-6mo) and the baseline baPWV. On the other hand, the determinants for the change of haPWV at 6 months (ΔhaPWV0-6mo) were baseline haPWV, ΔDBP0-6mo and the change of log-transformed plasma renin activity (ΔLogPRA0-6mo) The arterial stiffness of APA patients could be reversed after surgical treatment in the first 6 months after adrenalectomy. The determinant factors of baseline PWV included age, duration of hypertension and baseline SBP, and baseline DBP (for haPWV). The determinant factors of ΔPWV are baseline PWV, ΔDBP0-6mo and ΔLogPRA0-6mo (for ΔhaPWV). Since higher PWV was closely correlated with higher cardiovascular risks, the decrease in PWV might also imply reducing CV disease risks. However, we needed a further large scale clinical trial focusing on the long-term outcomes to clarify this.

碩士
國立臺灣大學
健康政策與管理研究所
99
Background：Primary aldosteronism is the most common cause of secondary hypertension. The two main causes of primary aldosteronism are aldosterone-producing adenoma, which could be cured by surgical intervention, and bilateral adrenal hyperplasia, which should be medical-treated. Computed tomography, adrenal venous sampling and adrenal scintigraphy are usually used for the etiology and lateralization of primary aldosteronism. Computed tomography is based on the anatomical findings, but it has wide variation of sensitivity. Adenal venous sampling is the gold standard for latearalization, but it is invasive and highly technical-dependent. Adrenal scintigraphy has limited role in primary aldosteronism during early days due to resolution. With the SPECT and SPECT/CT fusion imaging techique, the accuracy of adrenal scintigraphy is significant improved. The clinical algorithm for these modalities has not been well-established, and there are no studies concerning about the cost-effectiveness. Objective：This study aimed on the comparison of the accuracy, average cost and cost-effectiveness analysis of four imaging modalities for differentiating the etiology of primary aldosteronism, including CT, NP-59 planar, NP-59 SPECT and NP-59 SPECT/CT. Methods：We retrospectively included 27 clinical-confirmed primary aldosteronism patients from TAIPAI database. All of them had CT, NP-59 adrenal scintigraphy (plaran, SPECT and SPECT/CT) studies and pathology reports of adrenal lesion. The accuracy and average cost of these four imaging modalities were analyzed. By decision tress and setting effectiveness as diagnostic accuracy and diagnostic utility, cost-effectiveness were also analyzed. Sensitivity analysis were performed to determine the impact on the average cost while the variables changed. Results：NP-59 SPECT/CT is the most accurate study among four imaging modalities, with borderline significance as compared with NP-59 planar and CT. NP-59 SPECT/CT bares the lowest average cost, and NP-59 planar bares the highest average cost. By setting diagnostic accuracy as the effectiveness, NP-59 SPECT/CT has the lowest cost-effectiveness ratio. If setting diagnostic utility as the effectiveness, NP-59 planar is considered a ineffectiveness study. In sensitivity analysis, if the sensitivity of NP-59 SPECT/CT decreases to 0%, the average cost of NP-59 SPECT/CT is still lower the average cost of CT. The average cost of NP-59 SPECT/CT is higher than the average cost of CT while the specificity of NP-59 SPECT/CT decreases to 59.5%. Conclusion：Our study suggests that NP-59 SPECT/CT is the most accurate, the lowest average cost and the most cost-effective study among these four imaging modalities in differentiating the etiology of primary aldosteronism. It shows no significant impact on cost-effectiveness ratio as compared with CT while the sensitivity and specificity of NP-59 SPECT/CT changes. Aside from clinical concern, this result provides physicians consider the decision in another way to provide more effective management in primary aldosteronism.

The present thesis is a collection of the main research studies concerning imaging in PA, conducted during the PhD Course. In the second and third chapters, starting from the large cohort of PA patients recruited in the AVIS-2 study, we investigated respectively: a) if imaging by CT and/or MR could permit an accurate detection of PA and identification of unilateral PA; b) if a unilateral hypodense adrenal nodule alongside a contralateral normally appearing adrenal gland at imaging, can allow an accurate identification of unilateral surgically curable PA in patients aged 45 years or younger. In the fourth chapter I discussed the limitation of imaging in the diagnosis of PA and in its subtyping and which are the new possible algorithms and applications of imaging in PA, starting from the results obtained in the studies shown.

碩士
雲林科技大學
工業工程與管理研究所碩士班
96
Typical hypertension and Primary aldosteronism (PA) are the top two reasons for hypertension. Primary aldosteronism affects 5-15% of patients with hypertension. A PA patient could have hypertension and hypokalemia, but the study found that a PA patient may not have hypokalemia. It needs more indicators to diagnose PA. Typical hypertension and PA were often misjudged. This study applied Logistic regression analysis, C5.0, CART and Back-propagation Network to compare the prediction accuracy of PA. The best model is Logistic regression analysis. The Logistic regression analysis model had 85.9% accuracy for training and 91.9% accuracy for testing, and found six factors for predicting：HTNys、BMI、K、PACa、PRAa、MDRDchinese. The model of this study can improve the prediction for patient outcome (Typical hypertension or Primary aldosteronism) for clinical treatment. This study will help decrease the malpractice and the pain of patients.

Hypertension is a major risk factor for cardiovascular (CV) disease, and patients with primary aldosteronism (PA) - the most common endocrine cause of hypertension - have a higher incidence of CV complications. The aim of this study was to evaluate the incidence of metabolic differences and organ complications - kidney, heart and blood vessels damage in patients with essential hypertension (EH), PA and its most common forms - idiopathic hyperaldosteronism (IHA) and aldosterone-producing adenoma (APA). We found a higher incidence of metabolic syndrome and a higher incidence of metabolic abnormalities in IHA compared to APA - higher prevalence of metabolic syndrome, higher levels of triglycerides and lower levels of HDL cholesterol and thereby a higher cardiometabolic risk. Metabolic profile of patients with IHA is similar to EH in contrast to APA. Arterial stiffness was expressed as pulse wave velocity (PWV), in central arteries as carotid-femoral PWV and at peripheral level as femoral-ankle PWV. Patients with PA with comparable levels of blood pressure (BP) have higher stiffness of central elastic and peripheral muscular arteries than patients with EH. The main predictor of impaired peripheral arterial stiffness is the plasma aldosterone level. Patients with IHA have higher central arterial...

Myocardial damage is one of the most serious consequences of arterial hypertension. Changes in the heart structure and function develop not only due to pressure overload itself, but many other hemodynamic and neurohumoral factors contribute to their formation. Our work has compared echocardiohraphic strucutural anf functional changes of the left ventricle, caused by essential hypertension and hypertension associated with primary aldosteronism (PA) as the most common reason for secondary hypertension. The first part of our work focused on the differences in left ventricle geometry in men with PA and essential hypertension after separating it's low-renin form (where, similarly to PA, the plasma volume expansion was considered to have the dominant effect on left ventricle remodelation). In men with low-renin forms of hypertension including PA, we observed greater both endsystolic and enddiastolic diameter of the left ventricle, lower relative wall thickness and more frequent eccentric type of hypertrophy when compared to essential hypertensives with normal renin levels. Whereas left ventricle cavity diameters were positively correlated to aldosterone levels, wall thicknesses were associated mainly with hypertension severity expressed as an average 24hour blood pressure and number of antihypertensives....

Arterial stiffness represented by carotid-femoral pulse wave velocity (PWV) is considered to be an independent cardiovascular risk factor. This study was focused on large artery properties investigation in specific forms of hypertension using applanation tonometer Sphygmocor (Atcor Medical). PWV was significantly higher in resistant hypertension patients when compared to moderate essential hypertension (EH) patients. This difference appears to be independent of clinical blood pressure (BP). Night-time BP appears to be a more accurate predictor of PWV in EH. In another study we demonstrated that primary hyperparathyroidism (PH) (both hypertensive or non-hypertensive forms) might be associated with higher PWV when compared to EH patients or to normotensive controls and that this difference is independent of age and clinical BP. Neither calcium serum level, nor parathyroid hormone has been associated with PWV. Specific treatment by parathyroidectomy (PTX) seems to be beneficial for PWV decrease, which might be mainly determined by improved BP control after surgery. Since PTX indications for asymptomatic forms of PH have been discussed, our data suggest the potential benefit to the extent of subclinical organ damage after surgical treatment in these patients. Similarly, we prooved higher PWV in...

Les tumeurs du cortex surrénalien sont variées et fréquentes dans la population. Bien que des mutations aient été identifiées dans certains syndromes familiaux, les causes génétiques menant à la formation de tumeur du cortex surrénalien ne sont encore que peu connues. Un sous-type de ces tumeurs incluent les hyperplasies macronodulaires et sont pressenties comme la voie d’entrée de la tumorigenèse du cortex surrénalien. L’événement génétique le plus fréquemment observé dans ces tumeurs est l’expression aberrante d’un ou plusieurs récepteurs couplés aux protéines G qui contrôle la production de stéroïdes ainsi que la prolifération cellulaire. L’événement génétique menant à l’expression aberrante de ces récepteurs est encore inconnu. En utilisant le récepteur au peptide insulinotropique dépendant du glucose (GIP) comme modèle, cette étude se propose d’identifier les mécanismes moléculaires impliqués dans l’expression aberrante du récepteur au GIP (GIPR) dans les tumeurs du cortex surrénalien. Une partie clinique de cette étude se penchera sur l’identification de nouveaux cas de tumeurs surrénaliennes exprimant le GIPR de façon aberrante. Les patients étudiés seront soumis à un protocole d’investigation in vivo complet et les tumeurs prélevées seront étudiées extensivement in vitro par RT-PCR en temps réel, culture primaire des tumeurs, immunohistochimie et biopuces. Le lien entre le GIP et la physiologie normal sera également étudiée de cette façon. Une autre partie de l’étude utilisera les nouvelles techniques d’investigation à grande échelle en identifiant le transcriptome de différents cas de tumeurs exprimant le GIPR de façon aberrante. L’importance fonctionnelle des gènes identifiée par ces techniques sera confirmée dans des modèles cellulaires. Cette étude présente pour la première des cas de tumeurs productrices d’aldostérone présentant des réponses aberrantes, auparavant confinées aux tumeurs productrice de cortisol ou d’androgènes surrénaliens. Le cas probant présenté avait une production d’aldostérone sensible au GIP, le GIPR était surexprimé au niveau de l’ARNm et un fort marquage a été identifié dans la tumeur spécifiquement. Dans les surrénales normales, cette étude démontre que le GIP est impliqué dans le contrôle de la production d’aldostérone. Ces résultats ont été confirmés in vitro. Finalement, le profilage à grande échelle des niveaux d’expression de tous les gènes du génome a permis d’isoler une liste de gènes spécifiquement liés à la présence du GIPR dans des hyperplasies du cortex surrénalien. Cette liste inclus la périlipine, une protéine de stockage des lipides dans les adipocytes et la glande surrénale, dont l’expression est fortement réprimée dans les cas GIP-dépendant. Des études dans un modèle cellulaire démontrent que la répression de ce gène par siRNA est suffisante pour induire l’expression du récepteur au GIP et que cette protéine est impliquée dans la stimulation de la stéroïdogénèse par le GIP. En alliant des méthodes d’investigation in vivo de pointe à des techniques in vitro avancée, cette étude offre de nouveaux regards sur les liens entre le GIP et la physiologie de la glande surrénale, que ce soit dans des conditions normales ou pathologiques.
Tumors of the adrenal cortex are varied and frequently found in the population. Aside from rare family cases in which mutations have been identified, the genetic events leading to the formation of adrenocortical tumors remain obscure. A subtype of these tumors includes macronodular hyperplasias, now percieved as the entry point of adrenocortical tumorigenesis. The most commonly observed molecular event in these cases is the presence of aberrantly expressed G-protein coupled receptor that drive steroid production and cellular proliferation. The genetic events leading to these aberrant levels of expression are unknown. This study will use the Glucose-dependent Insulinotropic Polypeptide (GIP) receptor as a model to identify the molecular mecanisms leading to the aberrant expression of the GIP receptor (GIPR) in adrenocortical tumors. The first part of the study will be a clinical investigation of new cases of adrenocortical tumors to screen for aberrant responses to GIP in various types of these tumors. The patients will be evaluated by a thorough clinical investigation protocol and the resected tumors will be extensively analysed in vitro, using real-time RT-PCR, immunohistochemistry, microarray and primary cultures of the tumors. The link between GIP and the normal physiology of the adrenal cortex will also be assessed in normal subjects. The second part of the study will use novel large-scale investigation techniques to determine the transcriptome of different cases of adrenocortical tumors expressing aberrant levels of the GIPR. The functional importance of identified genes will be assessed in cellular models. This study presents the first cases of aldosterone-producing tumors with aberrant responses to hormones, previously confined to cortisol- or androgen producing tumors. The case presented showed an aldosterone production sensitive, among others, to GIP. The GIPR’s mRNA was strongly over expressed and a specific staining was observed in immunohistochemistry. The responses were confirmed in primary cultures of the tumor. In normal adrenals, a role for the control of aldosterone by GIP was also demonstrated. Finally, large-scale profiling of the transcriptome led to the identification of a list of genes with expression levels strictly related to the presence of the GIPR in adrenocortical hyperplasias. One of these genes, perilipin, was strongly repressed specifically in GIP-dependent cases. siRNA techniques were used in a cellular model and confirmed that the repression of perilipin is sufficient to induce the expression of GIPR and that this protein is implicated in the GIP induced steroidogenesis. Allying state-of-the-art in vivo investigation methods to advanced in vitro techniques, the present study identifies novel insights on the link between GIP and the normal adrenal physiology, in normal and pathological conditions.

博士
國立臺灣大學
臨床醫學研究所
100
Growing evidence has shown that high plasma aldosterone level leads to a risk of cardiovascular diseases (CVD), including fatal stroke and sudden cardiac death. In addition, long-term exposure to increased aldosterone levels resulted in renal and metabolic sequelae independently of the blood pressure level. This phenomenon is best demonstrated in patients with primary aldosteronism (PA) who have excessive and inappropriate production of aldosterone. The excessive production of aldosterone in PA is associated with a high incidence of cardiovascular events, in comparison with essential hypertension (EH). A total of 113 PA patients (87 patients with a diagnosis of aldosterone-producing adenoma, 26 with idiopathic hyperaldosteronism) and 55 patients with EH were enrolled. PA patients had higher arterial stiffness than EH patients (p = 0.006). However, lower numbers of circulating EPC and endothelial colony forming units (CFU) were observed in PA patients than in EH patients (p<0.05), which was ameliorated at six months after adrenalectomy or treatment with spironolactone. Expression of MR was identified in EPC. The plasma aldosterone concentration was inversely correlated with the number of circulating EPC (p =0.021) by the general additive model. The circulating number of EPC was inversely correlated with arterial stiffness (p=0.029) and the Framingham risk score (p=0.001). Serum high sensitively C-reactive protein was inversely correlated with the circulating EPC number (p=0.03) and decreased in patients after operation. Among the 45 patients who went through unilateral adrenalectomy, 32 (71%) were cured of hypertension. Preoperative number of circulation of EPC (Log[EPC number%] > -3.6) could predicted the curability of hypertension after adrenalectomy. (p=0.003). There was a biphasic effect of aldosterone on EPC proliferation. Incubation of early EPC with a low aldosterone level (10-9 and 10-8 M) for 5 days significantly increased the number of early EPC. However, higher dosage of aldosterone (10-6M) decreased the EPC number. EPC also showed a biphasic response to aldosterone, i.e., an increase of cell proliferation at low aldosterone level (10-9 and 10-8M), and a decrease at high aldosterone level (10-5 and 10-6M). Both effects were blunted by adding spironolactone. Aldosterone, in either a low or high dose, did not affect the percentage of senescence-associated β-galactosidase-positive EPC or apoptosis of EPC. After 5 days’ incubation with 10-6 M aldosterone, the functional capacity for tube formation of late EPC was significantly reduced (p<0.01). In contrast, a low dosages of aldosterone (10-8~10-9M) increased tube formation. This effect of aldosterone was dose-dependent (average total length and nodes number, all < 0.001). Our study clearly showed that human EPC express MR both at gene and protein levels. The expression of MR and 11β-HSD2 in EPC, therefore, indicates that EPC have the capacity to modulate gene expression in response to aldosterone specifically. In accordance with the animal experiments, our study reveals a long-term in vivo effect of high aldosterone level on the number of circulating EPC in PA patients, with an inverse association between PAC and EPC numbers. A correlation of PAC with oxidative stress and endothelial inflammation was observed in PA patients, and the oxidative stress was attenuated after adrenalectomy. Increased intracellular ROS may result in EPC mobilization and impaired neovascularization capacity. Accordingly, CRP augments the effect of aldosterone on endothelial cell stiffness. In our study, a significant correlation between CRP level and aldosterone was noted in PA patients. Moreover, there was an inverse correlation between CRP and EPC numbers, and a reduction of CRP level accompanied by an increase of EPC number was noted in APA patients after adrenalectomy. Chronic aldosterone excess leads to vascular morphological change (wall thickening and carotid artery fibrosis) and vascular dysfunction (central stiffness), independent of blood pressure (BP). In the present study, we further demonstrate that the lower the preoperative EPC number, the higher the risk of residual hypertension after adrenalectomy. The finding indicates that EPC play an important role in both normal endothelial function and vasculature. The inverse correlation between PAC and EPC number and the change of EPC numbers in response to the treatment of PA indicates that aldosterone contributes to the decreased EPC number in PA patients. The mechanisms accounting for low EPC numbers in PA patients are the direct activation of MR on EPC proliferation and an indirect effect through a high CRP level and oxidative stress caused by an excess of aldosterone. Low EPC numbers in PA plays a crucial role in the high incidence of arterial stiffness and in predicting residual hypertension in APA patients after adrenalectomy.

Der primäre Hyperaldosteronismus ist eine der häufigsten Ursachen einer sekundären Hypertonie. Entscheidend für eine adäquate Therapie ist die Subtypen-Differenzierung zwischen Aldosteron produzierenden Adenomen und bilateralen Hyperplasien der Nebennierenrinde. Hierfür wird heutzutage eine selektive Katheterisierung der Nebennierenvenen durchgeführt. Diese Untersuchung ist jedoch sehr aufwendig und geht mit einer hohen Strahlenbelastung einher. Daher wurden im Rahmen dieser Arbeit 11 fluorierte Inhibitoren der Aldosteronsynthase (CYP11B2) auf ihre in-vitro- und in-vivo-Wirksamkeit und Selektivität untersucht. Ziel war es, über eine gezielte Bindung radiofluorierter Tracer an Aldosteron produzierenden Zellen eine nicht invasive Bildgebung zur Subtypendifferenzierung beim primären Hyperaldosteronismus zu entwickeln. Hierbei konnten mehrere vielversprechende Substanzen gefunden werden, die selektiv an CYP11B2 binden und die Aldosteronproduktion hemmen. Diese können somit möglicherweise für die Subtypendifferenzierung beim primären Hyperaldosteronismus eingesetzt werden
Primary aldosteronism is one of the most common causes for secondary hypertension. The subtype differentation between aldosterone producing adenomas and bilateral adrenocortical hyperplasia is essential for a suitable treatment. Currently this affords a selective catherization of the adrenal veins. This test however is highly challenging and is attended by high radiation exposure. Therefore we analyzed the in vitro and in vivo effectivitiy of 11 fluorated aldosterone synthase (CYP11B2) inhibitors. The purpose was to develop a targeted imaging method with radiofluorinated tracers for the subtype differentation in primary aldosteronism. Several promising substances with a selective bond to CYP11B2 and inhibition of aldosterone production were found. These can possibly be used to differentate between the subtypes in primary aldosteronism