Architecture of the Distal Nephron Mineralocorticoid Receptor‐Dependent Transcriptome Defined
Authors: Jung Hyun Jun , Su Xiao-Tong , Al-Qusairi Lama , Ellison David H. , Welling Paul A. 
Published: April 2020

The mineralocorticoid receptor (MR), encoded by Nr3c2, is responsible for aldosterone‐regulation of Na+ and K+ balance and blood pressure. Although a handful of aldosterone/MR‐dependent genes have been identified, their regulation cannot fully explain how aldosterone activates electrogenic sodium‐potassium exchange in the aldosterone sensitive distal nephron (ASDN). Here, we apply next‐generation sequencing and systems biology approaches in isolated tubule segments of MR KO vs. Control mice to define a more complete inventory of MR‐dependent genes.

MRfl/fl/Pax8_rtTA/LC1 mice were used as a doxycycline (DOX)‐inducible Nr3c2 gene knockout model. After DOX treatment (14 days treatment, 7 days withdraw), four groups were prepared to distinguish between potassium and MR effects: 1) control mice on normal K+ diet (CT‐NK) or 2) high K+ diet (CT‐HK) and 3) MR knockout mice on normal K+ diet (KO‐NK) or 4) low K+ diet (KO‐LK). Transcriptome profiling using RNA sequencing (RNA‐Seq) was carried out in the micro‐dissected connecting tubule (CNT) and cortical collecting duct (CCD) tubule segments. For RNA‐Seq analysis, 5–6 mice per group and ~10 fresh ASDN tubules per mouse were prepared. cDNA libraries were sequenced on Illumina HiSeq4000 platform (2×75bp, paired end). Read count of each gene was calculated using Salmon and differential expression (DE) genes were identified using edgeR. Further downstream pathways analyses were performed using DE genes (FDR < 0.05).

DE analysis of transcriptome profiles identified 927 and 2,010 DE genes (FDR < 0.05) from comparisons of MR KO‐NK vs. CT‐NK and MR KO‐LK vs. CT‐HK, respectively. Diet effects were not detected. Absence of transcripts on the third exon of Nr3c2 gene confirmed complete disruption of Nr3c2 gene in the MR KO. All known aldosterone‐response genes, including Sgk1, Scnn1a, Ndrg2, Per3, Tsc22d3, Zbtb16, MLPH and Atp1a1 were significantly decreased in MR KO mice (low K+) compared to control mice (high K+). In addition, 5 DE genes (Sgk1, Scnn1a, Nedd4l, Fxyd4 and Atp1a1) were mapped on small known “Aldosterone‐regulated sodium reabsorption” profile in the KEGG database. However, genome‐wide identification of GR and MR binding sites using computational methods revealed that 526 of the significantly down‐regulated genes in MR KO mice are potential MR‐regulated genes, suggesting the aldosterone, MR‐dependent gene profile is much larger. Pathway enrichment analysis of 2,010 DE genes using multiple algorithms, GSEA (Gene Set Enrichment Analysis), KEGG, and Gene Ontology, consistently showed that DE genes were highly enriched in mitochondria‐associated metabolic processes, such as oxidative phosphorylation.

In conclusion, the inventory of MR‐regulated genes in the ASDN is much larger than previously imagined. In addition to pathways that directly up‐regulate epithelial sodium channel (ENaC) and the Na+‐K+ ATPase, the data suggest that aldosterone‐MR may directly influence metabolism to make energy‐consuming transport highly efficient.

Support or Funding Information

This study was supported by The LeDucq Foundation "Potassium in Hypertension".