**1. Introduction**

Primary aldosteronism (PA), affecting 6% of the general hypertensive population [1], and up to 20% of patients referred to hypertension units [2,3], is widely recognized as the leading cause of endocrine hypertension. Aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia (BAH) are the most frequent underlying causes of PA, while unilateral adrenal hyperplasia (UAH) is less common. The last few years witnessed major advances in the understanding of the molecular determinants leading to autonomous aldosterone overproduction in both sporadic and familial

PA. In particular, the introduction of next-generation sequencing allowed the identification of somatic mutations in four genes differently involved in Ca2+ homeostasis (*KCNJ5*, *ATP1A1*, *ATP2B3*, and *CACNA1D*), unraveling the genetic basis of approximately 50% of sporadic APAs [4–7]. Similarly, new insight was gained from mice lacking the core-clock components, cryptochrome-1 (CRY1) and cryptochrome-2 (CRY2) (*Cry*-null mice) [8]. Mammals, as well as many other organisms including plants, adapt most of their physiologic processes to a 24-h time cycle, generated by an internal molecular oscillator referred to as the circadian clock [9]. At the cellular level, circadian oscillations are generated by a series of genes, whose proteic products form a transcriptional autoregulatory feedback loop, where clock circadian regulator (CLOCK) and aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL, also known as BMAL1) act as positive regulators, while period (PER) and CRY act as negative regulators [10]. *Cry*-null mice displayed salt-sensitive hypertension due to chronic and autonomous aldosterone overproduction by the adrenal glands, as a consequence of the massive upregulation of type VI 3β-hydroxyl-steroid dehydrogenase (*Hsd3b6*), the murine counterpart to the human type I 3β-hydroxyl-steroid dehydrogenase (*HSD3B1*) gene [8]. HSD3B catalyzes the conversion of pregnenolone to progesterone, an enzymatic reaction required for aldosterone biosynthesis [11]; two different *HSD3B* isoforms are expressed in man—*HSD3B1* is mainly expressed in the placenta, while *HSD3B2* localizes primarily in adrenals and gonads [12]. Immunohistochemistry studies in normal human adrenals showed that *HSD3B2* is the predominant isoform, expressed through the zona glomerulosa and the zona fasciculata (ZF), while *HSD3B1* displays faint immunoreactivity, predominantly in the outermost layer zona glomerulosa (ZG) [8,13,14]. Moreover, in APA samples, *HSD3B1* expression was significantly correlated with the expression of the rate-limiting enzyme for aldosterone production—aldosterone synthase (CYP11B2) [15]. Despite much knowledge being gained from the *Cry*-null animal model, the significance of CRY1 and CRY2 in human adrenal function and aldosterone production is still unknown. So far, few reports have investigated the roles of *HSD3B1* and *HSD3B2* in sporadic PA. Therefore, in this study we aimed to (I) evaluate the expressions of *HSD3B1* and *HSD3B2* in a large cohort of 46 adrenal glands, removed from patients in whom a final diagnosis of unilateral PA was achieved; and (II) investigate the expression of *CRY1* and *CRY2* in unilateral sporadic PA, and their roles in aldosterone production in the HAC15 human adrenocortical cell model.
