Abstract 046: Deep Sequencing in mpkCCD Collecting Duct Cells Identifies Transcriptional Response to Vasopressin

Abstract

Blood pressure regulation depends in part on control of ion and water transport in the renal collecting duct by vasopressin and other hormones. To identify genes that are transcriptionally regulated by vasopressin in cultured mouse collecting duct cells (mpkCCD), we carried out both RNA-seq (n=9) and ChIP-seq for RNA polymerase II (Pol-II, n=3) after 24-hr treatment with vasopressin analog dDAVP or vehicle (Illumina HiSeq2000 sequencer). RNA-seq analysis showed significant changes in 33 transcripts out of 8393 quantified (Volcano plot thresholds: p<0.05 (Wilcoxon) and log2[ratio] outside of 99% confidence interval (CI) for vehicle-vs-vehicle controls). More transcripts increased (26) than decreased (7). The largest increases were seen for the transcriptional coactivator Cited1 (log2[dDAVP/Vehicle]= 5.2) and the water channel Aqp2 (log2[dDAVP/Vehicle]= 4.2). The ChIP-seq analysis showed significantly increased RNA-Pol-II binding in 209 genes versus only 9 with decreases. Plotting RNA-seq data versus ChIP-seq data for 3883 genes revealed 43 genes that exceeded the 95% CI for control-vs-control measurements for both variables, i.e., are transcriptionally regulated. The transcriptionally regulated genes included those involved in control of Na+/Cl- transport and/or blood pressure, viz. Fxyd4 (aldosterone-regulated channel-inducing factor), Tsc22d3 (the aldosterone-regulated leucine-zipper protein GILZ), Atp1b1 (Na+-K+-ATPase beta subunit) and Sik1 (salt-inducible kinase 1). Among transcriptionally upregulated genes were several transcription factors ( Elf3 , Nfkbiz , Nr4a1 , Myc ) that form the core of a proposed vasopressin/cAMP-activated transcriptional network. Conclusions: 1) Vasopressin signaling effects on transcription are biased with more genes increased than decreased. 2) Among the genes that are transcriptionally regulated in response to vasopressin are several that have been implicated in regulation of salt and water transport in the collecting duct.