Genetic mutations cause primary aldosteronism

Abstract

The human adrenal glands are complex endocrine organs that are physiologically located above the kidney. The cortex of the adrenal gland may be considered as a combination of three different steroidogenic tissue-types which form concentric zones within each adrenal. The three cortical zones include zona glomerulosa (ZG), zona fasciculata (ZF) and zona reticularis (ZR). Each zone, under independent regulation, produces unique steroid(s) which exhibit specific functions. The outermost ZG layer secretes the steroid, aldosterone due to ZG specific expression of aldosterone synthase (CYP11B2). Aldosterone regulates sodium reabsorption, and therefore, blood pressure. Aldosterone production is tightly regulated by the renin-angiotensin-aldosterone system. Thus, aldosterone levels are in direct proportion with renin levels. Other known physiological regulators of aldosterone production include serum K+ and adrenocorticotrophic hormone. A type of endocrine hypertension termed ‘Primary Aldosteronism’ (PA), is characterized by aldosterone secretion under suppressed renin levels. PA accounts for almost 10 % of hypertension. More recently, genetic mutations in an inward rectifying K+ channel (KCNJ5) that occur as both, somatic and germline cases, have been implicated in the pathology of PA. The goal of this dissertation is to define the role of KCNJ5 mutations in PA. In this dissertation, I will summarize my studies that describe the acute and chronic events involved in mutated KCNJ5 mediated aldosterone excess. In addition, I will define a novel mutation in KCNJ5 of germline nature identified at Georgia Regents University. Finally, I will also describe some interesting lessons we learnt from the expression of mutated KCNJ5 in primary cultures of human adrenals. The prevalence of a hereditary form of PA termed as Familial Hyperaldosteronism type III (FH III) is very rare. Thus far, only a few mutations in the KCNJ5 gene, including T158A, G151R, G151E and I157S, are confirmed as causing FH III, following Mendelian genetics. Perhaps the most interesting feature of this disease is the varied phenotype between the different mutations. T158A-affected patients present with massive hyperplasia and require bilateral adrenalectomy. In contrast, patients affected by the G151E mutation have more severe hypertension, although their adrenals are near normal in appearance. In this study we identify a new germline mutation (Y152C). The index case was a 61 year old woman who underwent unilateral adrenalectomy. The patient with the Y152C mutation exhibited a milder hypertension phenotype (like the G151E-affected patient) with extensive hyperplasia (as seen in the T158A-affected patient). In vitro analyses of the Y152C mutation indicated a pathology similar to other known mutations in KCNJ5, including change in conductance to Na+ ions and elevated calcium levels, and increase in CYP11B2 mRNA and aldosterone production. The inherent challenge presented by current studies utilizing constitutive expression of KCNJ5 mutations is the limitation in studying acute temporal events such as post translational modifications of steroidogenic enzymes and transcription factors. To address this issue, we generated a doxycycline inducible cell model system for the T158A harboring KCNJ5 transgene. Herein, we demonstrate a useful system that was amenable to the study of acute and chronic events involved in mutant-KCNJ5 mediated aldosterone excess. Our findings suggest that mutant KNCJ5 increases CYP11B2 expression through the activation of transcriptional activators of CYP11B2. Additionally, this is the first study to demonstrate that mutant KCNJ5 also activates steroidogenic acute regulatory protein (StAR) at the levels of translation and post translational phosphorylation. We also demonstrate calcium channel blocker, verapamil as an efficient blocker of mKCNJ5 mediated aldosterone production. Finally, one of the sharp advantages of our study was the use of primary cultures of human adrenal cells to confirm the effects of mutated KCNJ5. Interestingly, transduction of cells with constitutive viruses for mutant KCNJ5, confirmed an increase in KCNJ5 mRNA, although no change in CYP11B2 expression levels was observed. Pilot data including treatment of primary cells with calcium ionophores indicated that ZF/ZR cells may have a phenotype that is ‘muted’ for calcium mediated pathways. We could also speculate that this may disprove some current hypotheses that APA harboring KCNJ5 mutations may originate from the ZF. Overall, this study has improved our knowledge regarding the pathogenesis of PA caused by KCNJ5 mutations and has identified verapamil as a potentially effective therapeutic strategy in the inhibition of aldosterone excess in this type of PA.