Disordered CYP11B2 Expression in Primary Aldosteronism

Primary aldosteronism is the most common type of secondary hypertension affecting 6-10% of patients with primary hypertension. PA is mainly caused by unilateral hyperaldosteronism due to an aldosterone-producing adenoma, unilateral hyperplasia with or without micronodules or bilateral zona glomerulosa hyperplasias with or without macro or micronodules. The development of antibodies against the terminal enzyme of aldosterone biosynthesis (CYP11B2) has permitted the further characterization of normal adrenals and resected adrenals from patients with primary aldosteronism. Normal adrenals exhibit two different patterns of cellular expression of CYP11B2: young individuals display a relatively uniform expression of the enzyme throughout the zona glomerulosa while the adrenals of older individuals have dispersed CYP11B2-expressing cells but have more groups of cells called aldosterone-producing cell clusters (APCC). APAs exhibit different patterns of CYP11B2 staining that vary from uniform to homogeneous. There are also a proportion of cells within the APA that co-express different enzymes that are not normally co-expressed in normal individuals. Approximately 30% of patients with unilateral hyperaldosteronism do not have an APA, but either have an increased number of CYP11B2 expressing micronodules or hyperplasia of the zona glomerulosa. In summary, the studies reported in this review are shedding new light on the pathophysiology of primary aldosteronism. The wide variation in histopathological features of the adenomas and concurrent presence of APCCs raises the possibility that most cases of unilateral production of aldosterone actually might represent bilateral asymmetric hyperplasia with nodules frequently due to the development of somatic aldosterone-driving mutations

Polarized localization of phosphatidylserine in the endothelium regulates Kir2.1

Lipid regulation of ion channels is largely explored using in silico modeling with minimal experimentation in intact tissue; thus, the functional consequences of these predicted lipid-channel interactions within native cellular environments remain elusive. The goal of this study is to investigate how lipid regulation of endothelial Kir2.1 - an inwardly rectifying potassium channel that regulates membrane hyperpolarization - contributes to vasodilation in resistance arteries. First, we show that phosphatidylserine (PS) localizes to a specific subpopulation of myoendothelial junctions (MEJs), crucial signaling microdomains that regulate vasodilation in resistance arteries, and in silico data have implied that PS may compete with phosphatidylinositol 4,5-bisphosphate (PIP2) binding on Kir2.1. We found that Kir2.1-MEJs also contained PS, possibly indicating an interaction where PS regulates Kir2.1. Electrophysiology experiments on HEK cells demonstrate that PS blocks PIP2 activation of Kir2.1 and that addition of exogenous PS blocks PIP2-mediated Kir2.1 vasodilation in resistance arteries. Using a mouse model lacking canonical MEJs in resistance arteries (Elnfl/fl/Cdh5-Cre), PS localization in endothelium was disrupted and PIP2 activation of Kir2.1 was significantly increased. Taken together, our data suggest that PS enrichment to MEJs inhibits PIP2-mediated activation of Kir2.1 to tightly regulate changes in arterial diameter, and they demonstrate that the intracellular lipid localization within the endothelium is an important determinant of vascular function

Prevalence of KCNJ5 mutations and functional impact of a novel KCNJ5-insT149 mutation in aldosterone producing adenoma causing resistant hypertension

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

Prevalence of KCNJ5 mutations and functional impact of a novel KCNJ5-insT149 mutation in aldosterone producing adenoma causing resistant hypertension

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

Mutations of The Twik- Related Acid-Sensitive K+ Channel 2 (TASK-2) Promoter in Human Primary Aldosteronism

As a blunted expression of the twik-related acid-sensitive K+ channel 2 (TASK-2) is a common feature of aldosterone producing adenoma (APA) causing primary aldosteronism (PA), we sequenced the promoter region of the TASK-2 gene (KCNK5) in APAs (n=76), primary hypertensive patients (n=98) and 20 years-old healthy volunteers (n=71), searching for variants that could affect expression of this channel.We found TASK-2 promoter mutations in 24% of the APA: C999T in 6.6%, G595A in 5.3%, G36A in 5.3%, and C562T, Gins468, G265C, C1247T, G1140T and C1399T in 1.3% (each). The C999T mutation was found in only one of the 98 primary hypertensive patients, but mutations were detected also in 12% of volunteers: four carried the C999T, three G1288C, one the G1140T mutation and one the 468ins. After 16 years' follow-up none of these subjects developed hypertension or PA.The effect of C999T mutation was investigated in H295R cells using reporter vectors with the mutated or the wild-type (WT) TASK-2 promoters.TASK-2 gene expression was decreased by 31% (\ub118, p=0.01) in mutated, as compared to WT APA. Likewise, in transfected H295R cells, the C999T mutation decreased TASK-2 transcriptional activity by 35% (normalized luciferase signal fold change: 0.65\ub10.25 p<0.001).Thus, mutations in the promoter region of the TASK-2 gene can account for the low expression in about 24% of APA. As these mutations did not result into hypertension or PA during long-term follow-up in healthy subjects, they do not seem to be a factor causing PA by themselves