Part Ⅱ：The Pathology Of Aldosterone Biosynthesis And Its Action

The Pathology of Aldosterone Actions

Aldosterone actions in human lung and its disorders

We report for the first time the expression of 11β-HSD2 in the columnar epithelium of the respiratory fine bronchi of human lungs, co-localized with MR, which also suggests that aldosterone can be involved in the local or in situ regulation of sodium and water in human lungs. In rat models, aldosterone has also been reported to increase mRNA and protein levels of Na+, K+ -ATPase, and subsequently increase pulmonary edema clearance. In addition, a greater increase in 11β-HSD2 expression has been reported in fetal lung tissue compared to adult lungs to avoid the potentially deleterious effects of excess glucocorticoids and to further induce fluid retention in the airways to stimulate their growth. It is particularly interesting to note that aldosterone has also been reported to be involved in lung carcinogenesis. Glucocorticoids are known to inhibit lung tumorigenesis through a cyclooxygenase-2-mediated pathway. Thus, inhibition of 11β-HSD2 could have antitumor effects associated with increased tissue-active glucocorticoid levels and decreased COX-2 expression in lung tissue. Furthermore, RAAS blockers combined with systemic therapy for advanced non-small cell lung cancer improved the clinical outcomes of patients. Thus, aldosterone action as well as 11β-HSD2 may be considered as one of the poor prognostic factors in patients with lung cancer, and studies in this interesting area are currently underway. The Role of Aldosterone on the cardiovascular system and Its Disorders

Aldosterone was initially thought to regulate sodium and water homeostasis in the body mainly through its action in the kidneys. However, results from several studies have shown that aldosterone directly induces myocardial and vascular fibrosis, leading to increased morbidity and mortality, independent of systemic hypertension or abnormal electrolyte status. Aldosterone-dependent cardiac tissue fibrosis has been reported to be mediated by activation of NADPH oxidase, myocardial tissue production of reactive oxygen species (ROS) due to aldosterone action, and pro-inflammatory mediators. In a rat model of myocardial infarction, myocardial infarction resulted in a 2-fold increase in aldosterone synthase (CYP11B2) mRNA levels and a 3.7-fold increase in plasma aldosterone levels. In patients with congestive heart failure, elevated MR mRNA and protein levels were also detected in the left ventricle of the failing heart. Increased MR expression has also been reported in atrial tissue from patients with atrial fibrillation. In addition, 11β-HSD2 has been overexpressed in the left ventricle of rats with myocardial fibrosis and in atrial tissue from patients with atrial fibrillation. Of particular note, the mRNA levels of type 1 and type 3 collagen were significantly higher in stroke-prone spontaneously hypertensive rats than in control rats. In normal hearts, cardiomyocytes are constantly occupied by endogenous glucocorticoids due to relatively low levels of 11β-HSD2 expression. Thus, endogenous glucocorticoid-occupied MR may provide a protective effect relative to aldosterone-mediated MR activation, although there is controversy in this regard. In addition, treatment with the MR blocker spironolactone has been reported to significantly reduce the risk of death secondary to progressive hearing failure and sudden death in heart failure and to improve various symptoms of heart failure. This is mainly due to spironolactone-mediated potassium loss and the prevention of myocardial fibrosis.

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Aldosterone actions on vascular smooth muscle cells and their significance in vasculopathy

The presence of MR in the human vascular system was first reported in 1986 by Sasano et al. Subsequently, it was reported that MR is present not only in endothelial cells of the human vascular system but also in smooth muscle cells. Vascular structural remodeling has also been reported in small resistant medium-sized arteries in patients with primary aldosteronism compared with those with primary hypertension, suggesting that MR in smooth muscle cells can directly mediate the vascular remodeling process. We also previously demonstrated that aldosterone promotes the expression of MDM2, a nuclear protein involved in p53-mediated cell cycle prevention, which is subsequently attenuated by the aldosterone receptor blocker eplerenone. Furthermore, our in vivo results also showed that MDM2 expression was significantly higher in APA small artery smooth muscle cells than in nonfunctional adenomas and normal adrenal glands. Aldosterone-mediated vascular remodeling has also been reported in smooth muscle cells of MR knockout mouse models. In addition, MR-mediated pathways were also associated with 11β-HSD2 activity, albeit at low expression levels.MR has also been reported to be involved in SMC proliferation through Rho-kinase signaling, placental growth factor signaling, gal-3, and int-alpha 5 pathways. The above findings suggest that MR antagonists play a key role in preventing vascular remodeling and the development of further cardiovascular disease independent of plasma aldosterone levels.

Aldosterone Actions on chronic kidney disease

We previously reported that the prevalence of chronic kidney disease (CKD) in PA patients was significantly lower before PA treatment than after treatment throughout the 12-month follow-up period. Patients with aldosterone-producing adenoma and bilateral hyperaldosteronism (BHA) had an increased prevalence of CKD due to a reduced estimated glomerular filtration rate (eGFR), which reached approximately 20%. The above results also suggest a significant effect of PA on the pathogenesis of CKD. Elevated plasma aldosterone levels have been reported to be one of the major risk factors for the development of human kidney injury, which can be attenuated by MR antagonist therapy. For example, histologically confirmed renal damage in APA patients is more pronounced than expected from preoperative renal evaluation. MR activation can also cause renal endothelial dysfunction characterized by inflammatory activation, impaired vasodilation, and fibrosis. In addition, MR-mediated glomerulosclerosis can reduce capillary oxygenation capacity, ultimately leading to ischemic kidney injury. Spironolactone, one of the MR blockers, has also been reported to attenuate the decline in eGFR and the severity of histopathological lesions, ultimately protecting patients from potential ischemic kidney injury. Spironolactone has also been reported to exert renoprotective effects by reducing proteinuria. Eplerenone, another MR antagonist with attenuated effects in CKD, has been reported to be more selective for MR compared to spironolactone, which also binds to progesterone receptors. Eplerenone is a newly developed MR antagonist that is more selective for MR compared to spironolactone and eplerenone, and its anti-hypertensive effect has been demonstrated in in vitro studies. Results from phase III clinical trials of eplerenone have also demonstrated its efficacy not only in patients with hypertension but also in patients with type 2 diabetes and microalbuminuria, which could also make eplerenone a drug treatment option for patients with CKD.

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The pathology of primary Aldosteronism

Primary aldosteronism accounts for 5-10% of all hypertensive patients and is considered one of the most common endocrine disorders in this period. Considering the direct adverse effects of aldosterone on various tissues mentioned above, early detection and treatment of patients with PA is more critical than other hypertensive disorders. In addition, clarification of the pathological features of early or prodromal lesions in primary aldosteronism is essential to establish appropriate clinical management of patients with PA. This will also provide clinical benefit for patients who do not meet the classical criteria for PA, considering the systemic adverse effects of aldosterone excess independent of blood pressure and serum electrolyte levels, and the high frequency of PA in the general population. Therefore, we will discuss the latest developments in the pathology of primary aldosteronism in the next sections.

Somatic mutations in PA patients

A novel concept in PA pathology is that the vast majority of adrenocortical cells involved in autonomous aldosterone production and secretion have somatic mutations in ion channels or pumps, including potassium integer channel subfamily J member 5 (KCNJ5), calcium voltage-gated channel subunit α 1D (CACNA1D), ATPase Na+ /K+ transport subunit α 1 (ATP1A1), and ATPase plasma membrane Ca2 + transport 3 (ATP2B3), as previously described. The above results also suggest the significance or importance of these somatic mutations in PA pathology. In addition, the prevalence of somatic mutations in PA patients has been reported to depend on the race or ethnicity of the patient, and the clinicopathological and histopathological features differ between somatic mutations. For example, KCNJ5 mutations are the most common of all somatic mutations reported in PA patients, but they are also more common in East Asian APA patients, with a prevalence of almost 70% of all PA patients compared to 38% in Caucasian patients. On the other hand, ATP1A1 and CACNA1D mutations were more frequent in Caucasians than in East Asian PA patients.

Mutations in KCNJ5, encoding the inwardly rectifying K+ channel 4, lead to increased Na+ permeability, which in turn leads to sustained depolarization of the cell membrane of aldosterone-producing cells. This depolarized cell membrane induces cytoplasmic Ca2+ influx as a second messenger, which ultimately initiates aldosterone biosynthesis. mutations in CACNA1D encode the l-type calcium channel α-subunit CaV1.3, which regulates intracellular calcium homeostasis. mutations in the Na+ /K+ -ATPase α1 subunit ATP1A1 gene leads to calcium influx by altering sodium and potassium homeostasis. mutations in ATP2B3 directly affect the state of Ca2+ transport in the plasma membrane of the calcium pump, thereby increasing cytoplasmic calcium levels. All of these alter intracellular Ca levels, ultimately leading to excessive aldosterone production in mutant cells.

Somatic mutations in normal adrenal glands

Somatic mutations are present not only in APAs but also in APMs in normal adrenocortical ZGs. We previously reported the presence of somatic mutations in 21/61 APMs (34%) of normal adrenocortical ZG cells, including 14 CACNA1D mutations; 3 ATP2B3 mutations, 2 ATP1A1 mutations, and 2 simultaneous CACNA1D and ATP2B3 mutations. In normal adrenocortical ZG cells, there were 6 CACAN1D and 2 ATP1A1 mutations, and somatic mutations were also found in 8/23 APMs (35%). In addition, somatic mutations in KCNJ5 were detected in 5 APMs in the adrenocortical ZG adjacent to non-pathological APAs. On the other hand, aging has been reported as one of the potential factors for the development of APMs, since the number of APMs in normal ZGs increases with aging. This also suggests an association between aging and somatic mutations in related genes in the development of human adrenal APMs, but further studies are needed to clarify.

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The differentiation among APM, APN, and micro APA

An increasing number of PA cases are being diagnosed clinically through adrenal vein sampling, but most lesions are not always detected on routine CT imaging. These so-called "CT-negative lesions" include APDH, APN, APM, and aldosterone-producing micro APA, which must be confirmed by a detailed histopathological examination. As mentioned above, H&E-stained tissue sections can distinguish APN from APM histopathologically; however, micro APAs also exhibit histologic features similar to APN or APM and have been reported to be smaller than 10 mm in maximum size and only visible when examined on routine H&E-stained tissue sections. Therefore, definitive differentiation between APN/APM and micro-APA should be based on detailed histopathological analysis including validated CYP11B2 immunohistochemistry. A gradient of CYP11B2 immunoreactivity patterns from external to internal can be detected in non-tumorigenic APN /APMs, whereas the polarity of this unique CYP11B2 expression pattern can never be detected in tumorigenic micro-APAs.

Potential associations among APM, APN, and APA

With the detection of APM, associations between these aldosterone-producing lesions in each subtype have been reported recently. Indeed, no established transition pathway from one lesion to the other has been reported. However, our previously reported findings do show that both APN and APM share a similar somatic mutation profile, i.e., CACNA1D mutations were predominantly detected, although APM and APN were also not well distinguished at the time of analysis. The above results also suggest that there may be a non-tumorigenic transition pathway between APN and APM. However, whether non-tumorigenic APNs can be transformed into tumorigenic APA remains unknown, as the major KCNJ5 somatic mutation pattern of APAs is compared to the major CACNA1D somatic mutation pattern of APNs.

Morphology of aldosterone-producing adenoma

The normal adrenal cortex consists of ZG, ZF, and reticular zone (ZR) from the outside to the inside, but when normal adrenocortical cells differentiate or develop into nodules or tumors, including aldosterone-producing lesions, the cells of these lesions are usually a complex mixture of cells that mimic normal cortical cells, especially ZF and ZR cells. We have recently referred to these cells that mimic ZF and ZG/ZR cells as clear and compact cells rather than ZG or ZF-like cells to avoid potential confusion and possibly allude to the functional characteristics of the above-designated terms. Dense cells have a relatively high nucleus and poor cytoplasmic eosinophilic lipids. In contrast, clear cells have a relatively low nucleus-to-cytoplasm ratio and a lipid-rich cytoplasm. In APAs, we previously demonstrated that KCNJ5 mutant APAs consisted mainly of clear cells compared to KCNJ5 wild-type APAs. Furthermore, the relative intensity of CYP11B2 immune response was significantly and positively correlated with the number of clear cells, especially in KCNJ5 mutant APAs. On the other hand, we also demonstrated that ATP1A1 and CACNA1D mutant APAs consisted of more dense cells than clear cells. In contrast, ATP2B3 mutant APAs consisted mainly of hyaline tumor cells, to the same extent as KCNJ5 mutant APAs. In APMs or APNs, 13 of 32 nodules or micronodules consisted mainly of dense cells and 18 of 32 nodules consisted mainly of clear cells. The dominant genotype CACNA1D had a lower ratio of hyaline cells to dense cells compared to APA with the dominant genotype KCNJ5 mutation. aldosterone-producing lesions in APDH consisted of morphologically normal ZG cells, not dense or hyaline cells.

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Clinicopathological correlations of aldosterone-producing lesions

APA and BHA account for the vast majority of clinically overt PA cases, which does not usually pose diagnostic difficulties for most clinicians managing patients with PA. However, some patients with suppressed plasma renin activity but normal or slightly elevated plasma aldosterone levels have been reported to have normal blood pressure. APMs are usually detected in normal adrenocortical ZG, resulting in physiologic aldosterone production. However, APMs have also been reported to be found in normal ZG adjacent to APA lesions, suggesting that APMs are Autoren-independent. Furthermore, as mentioned above, APMs frequently undergo somatic mutations, and their number increases with age. The above findings suggest that physiological renin-dependent APMs may develop into pathological renin-dependent APMs due to aging or genetic disorders, which could also explain the normal blood pressure in PA. In addition, the area of APMs or APNs was larger in patients with IHA than in normal patients, suggesting that pathological APMs may also be associated with the change from normal to dominant PA, but further studies are needed to clarify this.

Xin Gao ¹. Yuto Yamazaki ¹, Yuta Tezuka ^2,3, Kei Omata ^2,3, Yoshikiyo Ono ³, Ryo Morimoto ³, Yasuhiro Nakamura ⁴, Takashi Suzuki ⁵, Fumitoshi Satoh ^2,3, and Hironobu Sasano ¹.

1. Department of Pathology, Tohoku University, Graduate School of Medicine, Sendai, Miyagi, Japan

2. Division of Clinical Hypertension, Endocrinology, and Metabolism, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan

3. Division of Nephrology, Endocrinology, and Vascular Medicine, Tohoku University Hospital, Sendai, Miyagi, Japan

4. Division of Pathology, Faculty of Medicine, Tohoku Medical and Pharmaceutical University, Sendai, Miyagi, Japan

5. Department of Pathology and Histotechnology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan