The Biology of Normal Zona Glomerulosa and Aldosterone-Producing Adenoma: Pathological Implications

Abstract

The identification of several germline and somatic ion channel mutations in aldosterone-producing adenomas (APAs) and detection of cell clusters that can be responsible for excess aldosterone production, as well as the isolation of autoantibodies activating the angiotensin II type 1 receptor, have rapidly advanced the understanding of the biology of primary aldosteronism (PA), particularly that of APA. Hence, the main purpose of this review is to discuss how discoveries of the last decade could affect histopathology analysis and clinical practice. The structural remodeling through development and aging of the human adrenal cortex, particularly of the zona glomerulosa, and the complex regulation of aldosterone, with emphasis on the concepts of zonation and channelopathies, will be addressed. Finally, the diagnostic workup for PA and its subtyping to optimize treatment are reviewed.

Figures

Figure 1.

Steroidogenic pathways in the human adrenal cortex. Enzymes localized in the mitochondria are in yellow boxes. [© 2018 Illustration Presentation ENDOCRINE SOCIETY]

Figure 2.

Ontogeny of the human adrenal and time during gestation at which steroidogenic genes are first expressed and steroids synthesized. CYB5R3, cytochrome b5 reductase; HSD3B, 3β-hydroxysteroid dehydrogenase; SULT1A, sulfotransferase. [© 2018 Illustration Presentation ENDOCRINE SOCIETY]

Figure 3.

Activation of wild-type and mutated channels. (a) Under physiological conditions the threshold of voltage-operated T-type calcium channels Cav3.2 (blue) is lower than that of l-type Cav1.3 channels (green), leading to activation (and deactivation) at lower voltages (permissive window of voltage), even though the voltage dependence [i.e., the fractional opening per unit increase in voltage (slope factor)] does not differ greatly between channels. The pink rectangle indicates the range of resting membrane potentials of normal glomerulosa cells. The y-axis indicates the amplitude of currents, expressed as normalized steady-state current. (b) Both somatic and germline KCNJ5 mutations cause loss of ion selectivity with ensuing increased Na+ influx and therefore a shift of the resting potential toward less polarized voltages (striped rectangle) that, in turn, causes opening of the voltage-dependent T-type Ca2+ channels [see Choi et al. (19)]. (c) Germline CLCN2 mutations, by causing efflux of chloride ion, also determine a shift of the resting potential toward less polarized voltages [see Scholl et al. (23) and Fernandes-Rosa et al. (24)]. (d) A similar shift of the resting potential toward less polarized voltages can occur in cells with mutations of ATP1A1 that cause loss of function or with mutations of ATP2B3 associated with impaired clearance of cytoplasmic Ca2+ ions [see Beuschlein et al. (138)]. (e) Ile770Met CACNA1D mutation (brown dotted line) causes a curve shift toward the left, leading to activation of the mutated channel at lower voltages than those needed to activate the wild-type Cav1.3 channel (green) [see Scholl et al. (139)]. (f) Mutations of CACNA, as germline CACNA1H M1549V mutation, cause increased constitutive Ca2+ influx at potentials close to the resting potential of zona glomerulosa cells (more rapid activation) and delayed inactivation, with a resulting larger permissive voltage window [see Scholl et al. (140)]. For details on cell type and species in which these curves were generated, see the original references. [© 2018 Illustration Presentation ENDOCRINE SOCIETY]

Figure 4.

Mechanisms mostly deemed to contribute to the development of APAs. (a) Somatic mutations in the genes KCNJ5, ATP1A1, and CLCN2 coding for Kir3.4, Na+/K+ ATPase, and ClC-2 channels cause membrane depolarization of zona glomerulosa, with ensuing increased influx of Ca2+ into the cells, whereas mutations in CACNA and ATP2B3 genes, which code for Cav1.3 and plasma membrane calcium-transporting ATPase, directly cause an increase in intracellular Ca2+ levels. In both cases the final result is enhanced CYP11B2 expression and increased aldosterone production. For a list of mutations, see Table 1. (b) Wnts are secreted proteins that control growth and stem cell renewal, acting via canonical and noncanonical Wnt pathways. The canonical Wnt signaling is activated by the binding of Wnt to a serpentine Frizzled receptor (Fzd) and the coreceptor LRP5/6, which lead to recruitment of Dishevelled protein Dsh and disassembly of the β-catenin destruction complex. β-catenin, free to move toward the nucleus, binds to the TCF/LEF1 proteins, triggering transcription of genes involved in growth. When activated by the noncanonical Wnt pathway, Wnt first binds to Fzd and recruits Dsh to form a complex that activates the Rho-associated kinase pathway or phospholipase C, finally releasing Ca2+ from intracellular stores. The increased Ca2+ can drive cell growth or aldosterone synthesis. (c) Hypomethylation of the promoter region of CYP11B2 activates gene transcription, thereby promoting aldosterone secretion. (d) Elevated levels of miRNA 23 (miR23) and miRNA 34 (miR34) can downregulate KCNJ5 gene expression, blunting TASK2 synthesis, and finally enhancing aldosterone production. [© 2018 Illustration Presentation ENDOCRINE SOCIETY]

Figure 5.

Development of APA or multinodular cortical hyperplasia from zona glomerulosa. In FH-I the hybrid chimeric gene comprising the CYP11B2 coding sequences under control of the CYP11B1 promoter induces hyperproduction of aldosterone controlled by ACTH. Growth and proliferation of stem cells cause multinodular cortical hyperplasia or multinodular lesions with tumors. In the only FH-I pedigree [see Jeunemaitre et al. (163) and Pascoe et al. (172)], two patients had a tumor but did not lateralize on adrenal venous sampling (AVS), suggesting that these tumors were not APAs. In FH-III the excess production of aldosterone is caused by mutations in the KCNJ5 gene, which are located in or near the selectivity filter. Whether growth and proliferation of stem cells cause development of APAs remains to be investigated. [© 2018 Illustration Presentation ENDOCRINE SOCIETY]

Figure 6.

Steroid synthesis in APA cells and origin of APA cells from zona glomerulosa or zona fasciculata. [(a), left] Staining of an APA with hematoxylin and eosin identifies zona glomerulosa–like cells (visualized as pink cells) and zona fasciculata–like cells (yellow). However, hematoxylin and eosin cannot provide functional information about steroidogenetic capability. Note the discontinuous layer of zona glomerulosa cells under the capsule. [(a), right] Immunohistochemistry with antibodies against CYP11B1 and CYP11B2 allows identification of cortisol-producing (green) and aldosterone-producing (violet) cells in the APA, as well as aldosterone-producing (violet) cells in the subcapsular clusters. Both CYP11B1- and CYP11B2-positive cells can be detected in the APA, suggesting that there is no perfect matching between zona fasciculata–like cells. Moreover, the APA may also contain CYP17 positive cells or even double (CYP17 and CYP11B2 or CYP17 and CYP11B1) or triple (CYP17 and CYP11B2 and CYP11B1) positive cells, intermingled with cells that do not show any immunoreaction. (b) Illustration of the two-hit theory with its variants. First, an injury or mutation (first hit) in a CYP11B2-positive cell of zona glomerulosa causes hyperproduction of aldosterone and transforms the zona glomerulosa cell into a zona fasciculata–like cell; then, activation of a pathway involved in cell growth (e.g., Wnt; second hit) causes abnormal proliferation with the tumor mass (APA). Second, the injury or mutation (first hit) occurs in a CYP11B1-positive cell of zona fasciculata, with transformation of the cell into a cell producing aldosterone; the second hit causes abnormal proliferation with the tumor mass (APA). Finally, the injury or mutation can associate with expression of other enzymes involved in steroidogenesis, as CYP17. To date there are no evidence supporting the superiority of one theory over the other. Note that the colors used to visualize the cells have been chosen arbitrarily. [© 2018 Illustration Presentation ENDOCRINE SOCIETY]