Document Type

Thesis

Date of Degree

2014

Degree Name

MS (Master of Science)

Degree In

Pharmacology

First Advisor

Curt D. Sigmund

Abstract

Renin (REN) expression is required to maintain blood pressure and electrolyte homeostasis. However, the mechanisms by which REN is transcriptionally regulated remain elusive. We reported a functional role for several nuclear receptors (NRs) on REN gene transcription. To identify other candidate NRs that regulate REN, we analyzed a publicly available microarray dataset (GUDMAP Developing Kidney ST1) to compare the expression pattern of REN and the 48 NRs across different kidney cell types. Our analysis revealed 14 NRs exhibiting a similar pattern as REN. We hypothesized that these NRs are co-regulated with REN and can regulate REN transcription. To test this hypothesis, we set up 2 cohorts of mice in which REN expression was either high or low compared to control mice and measured expression of REN and NRs in renal cortex by qPCR. The high-REN cohort was given the ACE inhibitor captopril (100g/kg/day) for 10 days, and the low-REN group was implanted subcutaneously with a deoxycorticosterone acetate pellet (50mg) and received 0.15 M NaCl in drinking water for 21 days (DOCA-salt) in addition to water. Captopril increased REN and reduced NR2F6 expression relative to vehicle treatment (REN: 16±1, p<0.01; NR2F6: 0.5±0.04, p<0.01). DOCA-salt reduced expression of REN and several NRs relative to vehicle (REN: 0.1±0.02, p<0.01; estrogen receptor alpha ESR1: 0.1±0.08, p<0.01; thyroid receptor beta: 0.1±0.03, p<0.01; vitamin D receptor: 0.2±0.04, p<0.001). We used REN-expressing As4.1 cells to investigate the molecular mechanisms by which one of the co-regulated NRs (ESR1) might directly regulate REN transcription. siRNA-mediated knockdown of ESR1 by 75% in As4.1 cells was associated with a 2-fold reduction of REN. Moreover, DNA affinity precipitation assays demonstrated the ability of ESR1 to bind to the hormone response element in the REN enhancer and chromatin immunoprecipitation indicated that ESR1 binding is enriched in the REN enhancer region (fold enrichment 29.1±6, p<0.05 vs IgG). Thus, by comparing gene expression profiles we identified candidate transcriptional regulators of REN. Experimental studies confirmed that ESR1 directly modulates REN transcription. Further studies are required to fully elucidate the mechanism by which ESR1 regulates REN.

Pages

ix, 68 pages

Bibliography

Includes bibliographical references (pages 50-62).

Copyright

Copyright 2014 Ko-Ting Lu

Included in

Pharmacology Commons

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