Characterization of Aldosterone-producing Cell Cluster (APCC) at Single-cell Resolution

Context: The adrenal cortex consists of zona glomerulosa (ZG), fasciculata (ZF), and reticularis. Aldosterone-producing cell clusters (APCCs) that strongly express aldosterone synthase (CYP11B2) are frequently found in adult adrenals and harbor somatic mutations that are also detected in aldosterone-producing adenomas (APAs). Primary aldosteronism is mainly caused by APAs or idiopathic hyperaldosteronism (IHA). We presume that APCCs are causing IHA and are precursors of APAs. However, the gene expression characteristics and especially the development of APCCs are not well understood. Objective: This study aimed to analyze the transcriptome of APCCs at single-cell resolution and infer the developmental trajectory. Methods: Single-cell RNA sequencing (scRNA-seq) of 2 adult adrenals was performed. Results: Immunohistochemical analyses confirmed the 2 adrenals had APCCs. scRNA-seq data of 2928 adrenal cells were obtained and 1765 adrenocortical cells were identified based on unsupervised clustering and the marker gene expression. The adrenocortical cells were divided into 6 clusters, of which 3 clusters (923 cells) were composed of APCC/ZG cells. By further subclustering, the APCC/ZG cells were divided into 3 clusters (clusters C1, C2, and C3), we finally identified APCC cluster (C3) and ZG cluster (C1). Cluster C2 seemed to be ZG-to-ZF transitional cells. RNA velocity analysis inferred the developmental direction from cluster ZG-cluster-C1 to APCC-cluster-C3. The scRNA-seq additionally revealed that many CYP11B2-positive cells were positive for CYP11B1 and/or CYP17A1, which were essential for cortisol but not for aldosterone production. Conclusions: Our results revealed the gene expression characteristics of APCC at single-cell resolution and show that some ZG cells remodel to APCC. Key Words: single-cell RNA sequencing, transcriptomic study, adrenal gland, aldosterone-producing cell cluster Abbreviations: APA, aldosterone-producing adenoma; APC, allophycocyanin; APCC, aldosterone-producing cell cluster; CCS, cosmic calf serum; CYP11B1, cytochrome P450 family 11 subfamily B member 1; CY11B2, cytochrome P450 family 11 subfamily B member 2; DEG, differentially expressed gene; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; GO, gene ontology; IHA, idiopathic hyperaldosteronism; PA, primary aldosteronism; scRNA-seq, single-cell RNA sequencing; UMAP, Uniform Manifold Approximation and Projection; ZF, zona fasciculata; ZG, ;zona glomeru