Date of Degree
PhD (Doctor of Philosophy)
Molecular Physiology and Biophysics
Curt D. Sigmund
The renin angiotensin system (RAS) is critical for the regulation of blood pressure, electrolyte/fluid, and metabolic homeostasis. Regulation of the RAS is important in the development and treatment of hypertension. As part of the rate-limiting step in a cascade of events ending in the production of angiotensin II, renin is a major regulator of the RAS. Its expression is localized to the juxtaglomerular (JG) cells of the JG apparatus where it is exquisitely located to respond to various physiological cues. Understanding the regulation of renin expression and development of the juxtaglomerular cells is critical. Two regulatory elements, the enhancer and proximal promoter, have been found to be important in controlling cell- and tissue- specific baseline expression of the renin gene. Within the enhancer is a hormone response element (HRE) which confers a high level of activity to the enhancer. Nuclear receptors that bind this element have been found to bind the HRE and regulate renin promoter transcriptional activity. We have previously characterized the role of the orphan nuclear receptor Nr2f6 as a negative regulator of renin expression that mediates its effects through the HRE. However, gel shift assays indicate that there are other transcription factors binding this element. We have identified other orphan nuclear receptors that regulate renin expression. The first, Nr2f2 acts as a negative regulator of renin promoter activity but does not appear to affect baseline expression of the endogenous renin gene. The other, Nr4a1, is a positive regulator of renin expression, but it does not appear to mediate its effects through the HRE.
The transcriptional regulation of gene expression is controlled by regulatory elements separated by large distances from promoters. We and others have found that short transgenes of the human renin (hREN) locus are not sufficient to protect them from positional effects that can be exerted upon them by neighboring regulatory elements. We discovered a random truncation in a large genomic construct of the hREN gene that resulted in ubiquitous expression of renin not seen with the intact form. By locating the genomic insertion site of that transgene in the Zbtb20 gene, we found that the hREN promoter had come under control of that gene's regulatory elements. The gene downstream of renin however maintained its tissue-specific expression. We found that CCCTC-binding factor (CTCF) bound to chromatin in and around the renin locus. The presence of CTCF suggests that insulator elements are present in the renin locus, and their loss likely explains the results above.
Finally, we assessed the role of microRNAs in the development of renin expressing cells in the mouse kidneys by cell-specific deletion of the processing enzyme Dicer. This resulted in reduction of renin expression and a decrease in the number of renin expressing cells in the kidney. Mice were hypotensive and had several kidney abnormalities including a hypertrophied vasculature and striped fibrosis. These results indicate that Dicer and the miRNAs it processes are critical for the development and maintenance of renin expressing cells that contribute to normal kidney development.
CTCF, Dicer, Nuclear Receptor, Renin, Transcription
viii, 96 pages
Includes bibliographical references (pages 81-96).
Copyright 2011 Eric Thomas Weatherford