CACNA1-D and KCNJ5-mutant aldosterone-producing adenomas (APAS) have opposite 2-year clinical outcomes from adrenalectomy: prospective trial findings explained by different cells-of-origin.

Abstract

In a recent trial, adrenalectomy achieved complete clinical success at 6 months in only 30% of 78 patients with unilateral primary aldosteronism (PA) (Nat Med 2023 29:190). We have now compared influence of common APA genotypes on success at 2 years, and sought different cells of origin to explain the result. Method(s): Home BP, plasma aldosterone and renin were re-measured 2 years after surgery. APA genotype and transcriptome were analysed by RNAseq. Single-nucleus (Sn) RNAs from 17 fresh-frozen adrenals and 9 APAs (3 each with CACNA1D or KCNJ5-mutation), were quantified by 10x Chromium, and cells with mutations identified by targeted amplicon analysis. Unusual cells were confirmed by immunofluorescence (IF), and their origin sought in published datasets for other tissues. Result(s): 14/18 patients with KCNJ5-mutation, but 0/20 with CACNA1D, had BP <135/85 mmHg off treatment at 2 years (p=0.03, logistic regression on genotype, age, gender, ethnicity). Biochemical success rate was also lower in CACNA1D-mutant patients. The most upregulated gene in CACNAID vs KCNJ5-mutant APAs was the endothelial-progenitor CCM2L (33-fold, p=10-26), followed by neuronal and adrenomedullary genes e.g. PTPRZ1, SLC35F1, UNC79, by 13-17-fold, p=10-[1]0-[1]2). Transcripts of these genes, in Sn analyses of 85203 nuclei from 17 non-tumour samples, clustered in CYP11B2- expressing cells, along with genes characteristic of aldosterone-producing micronodules (APM). TheAPMcluster was downstream of medulla on pseudotime analysis. In Sn analysis of 9 APAs, CCM2L was highly differentiated in CYP11B2+ cells from 3 CACNA1D- vs 3 KCNJ5-mutants (p=10-[1]06) along with PTPRZ1 (p=10-232), SLC35F1 (p=0) and many other neuronal/medullary transcripts. UMAPs of APAs and adjacent adrenal both revealed a cluster of hybrid cells expressing CCM2L or other endothelial genes, plus CYP11B2 and other zona glomerulosa (ZG) or APM-selective transcripts. The proportion of hybrid cells was higher in CACNA1D-mutant APAs (236/803) and adjacent adrenal (37/946) than KCNJ5-mutant (101/954) and adjacent tissue (8/766). By contrast, CYP11B2+ cells from KCNJ5-mutant APAs were enriched for transcripts in the CYP11B2- ZG-cell cluster of adjacent adrenal. Amplicon analysis of APAs detected CACNA1D mutation in multiple cells of the endothelial cluster. IF confirmed cells expressing CYP11B2 and endothelial transcripts. Mining of GTEX, and Human Cell Atlas led to a cluster of CD34+RENBP+ endothelial progenitors in spleen as likely source of CCM2L+ cells (Gen Biol 2019 21:1). Conclusion(s): Somatic genotype influences long-term outcome after adrenalectomy for PA, reflecting origin of CACNA1D-, but not KCNJ5-, mutant APAs from APMs. Bilateral APMs have been inferred from their occurrence in most PA adrenals (Nat Rev Neph 2023 19:788). This is supported by our finding of CCM2L+CYP11B2+ hybrid cells, with a likely circulatory origin from spleen.