A DIFFERENT PERSPECTIVE ON THE PATHOPHYSIOLOGY OF PRIMARY ALDOSTERONISM BY TRANSCRIPTOMIC ANALYSIS OF URINARY EXTRACELLULAR VESICLES

Objective: To compare the transcriptomic profiles of urinary extracellular vesicles (UEVs) collected from patients with Primary Aldosteronism (PA), specifically with Aldosterone-Producing Adenoma (APA) before and after surgery, and subjects with Essential Hypertension (EH). Design and method: UEVs were isolated using an ultrafiltration-based method and a commercially available precipitating reagent. RNA libraries were prepared according to the TruSeq Stranded Total RNA protocol and sequenced by NextSeq System (Illumina). A selected panel of differentially expressed genes (DEGs) was assayed for validation through droplet digital PCR (ddPCR). Results: Enrichment analysis of DEGs in 5 patients with APA, compared to 8 subjects with EH, revealed several significantly overrepresented transcript classes, including some related to ion transport, sodium channel activity, and extracellular matrix composition. Notably, seven genes encoding subunits of voltage-dependent calcium (CACNA2D1, CACNA2D4, CACNG2) and sodium (SCN3A, SCN5A, SCN9A, SCN11A) channels were upregulated in APA patients. Further analyses aimed to identify potential aldosterone-regulated genes, focusing on DEGs shared between the comparisons of APA vs. EH and APA pre- vs. post-surgery. Within this group, ten genes expressed in APA were identified as particularly noteworthy based on their molecular functions: CST3, EPHB2, PRDM5, NADK, HLA-DQA1, and ITGA5 for their roles in extracellular matrix remodeling, kidney fibrosis, and inflammatory responses; PIK3R2 for its involvement in aldosterone-regulated sodium reabsorption; CACNA2D4 and ABCC8 for ion transport; and ADD2 for its association with hypertension. RNA-Seq results were validated in an independent cohort (8 APA pre-surgery, 5 APA post-surgery, 8 EH) using ddPCR, selecting 18 of the most notable DEGs. Two genes (PIK3R2 and CST3) confirmed the trends observed by RNA-Seq, even if the differences were not statistically significant. Conclusions: This pilot study further corroborates the importance of ion channel dysregulation, extracellular matrix remodeling, and inflammation in the pathophysiology of PA. Additionally, our findings indicate that UEVs mRNA may reflect a wide range of processes occurring in urinary tract cells. Despite the challenges posed by the intrinsic variability of UEVs, these vesicles represent a valuable and accessible tool to explore the pathophysiological mechanisms occurring in the kidney, and useful for risk stratification in the context of PA.