UNIVERSITY OF CINCINNATI Date:___________________ I, _________________________________________________________, hereby submit this work as part of the requirements for the degree of: in: It is entitled: This work and its defense approved by: Chair: _______________________________ _______________________________ _______________________________ _______________________________ _______________________________ The Role of Uroguanylin in the Regulation of Renin Angiotensin Aldosterone System A thesis submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in the Molecular and Developmental Biology Program of the College of Medicine 2006 By Shams Tabrez Quazi M.B.,B.S., Pune University, India, 2003 Committee Chair: Katherine Yutzey, Ph.D. i Abstract Dysregulation of the Renin Angiotensin Aldosterone System (RAAS) is implicated in diseases such as hypertension and congestive heart failure. cGMP regulating systems work as physiological antagonists to counter harmful RAAS hyperactivity. Uroguanylin (UGN) is one such cGMP regulating peptide, but its effect on RAAS has not been studied. On the basis of background information from other cGMP regulating factors like atrial natriuretic peptide (ANP), as well as the phenotype of UGN knockout mice, we formulated the hypothesis that UGN negatively regulates renin expression and/or secretion. The microarray results were consistent with our hypothesis that UGN inhibits renin mRNA expression but we could not validate this by Real-Time PCR. A possible reason behind this discrepancy could be the remarkable effect of psychosocial stress on renin expression, a parameter not considered during our microarray experiment. Future studies could be designed to explore the possible negative role of UGN on renin secretion. ii iii Acknowledgements It is my pleasure to thank all the people that made this Master’s thesis possible and for making this endeavor an enjoyable one. I would like to acknowledge my thesis committee, Dr. Katherine Yutzey, Dr. Mitchell Cohen, and Dr. Kris Steinbrecher for their time and advice. I also thank the Molecular and Developmental Biology Graduate program for having provided me the opportunity to further my education. It is difficult to overstate my gratitude to my advisor, Dr. Mitchell Cohen. His appreciation and encouragement was invaluable during the two years I was associated with him and his lab. I saw in him an ideal mentor who displayed the correct balance between providing me with freedom to think on my own and also being there when I needed guidance. I will cherish his mentorship all my life. I would also like to thank everyone in Dr. Cohen’s lab for providing a great working environment. I appreciate all the advice and guidance I got from Dr. Kris Steinbrecher, Dr. Maksood Wani, and Dr. Elizabeth Mann. I thank Dr. Monica Garin-Laflam for being a friend and a colleague. I also thank Jen Hawkins and Juxian Mao for their technical expertise and support. iv Table of Contents Title i Abstract ii Blank Page iii Acknowledgement iv Table of Contents v Introduction 1 Background and Significance 6 Materials and Methods 14 Results 17 Discussion 26 References 30 v Introduction Guanylin (GN) and uroguanylin (UGN), also referred to as “guanylin peptides,” are small molecular weight (~12kD) proteins that are found naturally in humans and other mammals. These peptides are similar to the heat-stable toxin (ST) secreted by E.coli that causes traveler’s diarrhea (1). Identification of ST-producing organisms in large numbers of patients with E. coli diarrhea led to studies to find their mechanism of action. It was discovered that ST increased intracellular cGMP and subsequently, it was shown that the ST receptor was a transmembrane protein, guanylate cyclase C (GC-C) (2). GC-C was shown to be expressed at high levels in the apical membrane of the intestinal epithelium and eventually it was found to be expressed in various other organs such as kidney, regenerating liver, testes, placenta and uterus (3, 4). The search for endogenous ligands for this receptor led to the isolation of GN from embryonic rat intestine and a little later, of UGN from opossum urine (5, 6). Both peptides were shown to bind to and activate the GC-C receptor. These peptides are also found in various human and other mammalian organs including the intestinal epithelium, kidney, brain, placenta and the uterus and are secreted as inactive prohormones (4, 7). The guanylin peptides have significant homology to each other, both at the nucleotide level, and at primary protein sequence level (Table I). Amino terminus Carboxyl termi nus E.coli STporcine N-T-F-Y-C-C-E-L-C-C-N-P-A-C-A-G-C-Y E.coli SThuman N-S-S-N-Y-C-C-E-L-C-C-N-P-A-C-T-G-C-Y GNhuman P-G-T-C-E-I-C-A-Y-A-A-C-T-G-C GNmouse P-N-T-C-E-I-C-A-Y-A-A-C-T-G-C UGNhuman N-D-D-C-E-L-C-V-N-V-A-C-T-G-C-L UGNmouse T-D-E-C-E-L-C-I-N-V-A-C-T-G-C Table I. The guanylin peptide family. There is a large degree of homology at the protein level between guanylin, uroguanylin and ST and between peptides from different species. 1 After they are secreted in the inactive pro-hormone form, they are cleaved to produce the active form that binds to GC-C. The cysteine rich carboxy terminal is the active portion of ST and the guanylin peptides. Binding of this carboxy terminal to the extracellular domain of GC-C activates the intracellular guanylate cyclase domain raising intracellular cGMP levels as shown in Figure 1 (6). This in turn has multiple effects on intracellular kinases, phosphodiesterases and ion transporters. Perhaps the most studied effect is the activation of cGMP-dependent protein kinase II (cGK-II) (10). Activation of cGK-II causes the phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) and consequently results in bicarbonate and chloride ion secretion. Figure 1. Schematic representation of GN/UGN signaling cascade. GN, UGN and ST (also known as STa) bind to the extracellular domain of GC-C producing conformational changes in the intracellular cyclase domain that generates cGMP from GTP. Increased intracellular cGMP activates cGK-II that phosphorylates CFTR causing chloride and bicarbonate secretion. 2 Soon after the isolation of the UGN and GN peptides, their genes were localized to the human chromosome 1 and mouse chromosome 4 and were found to consist of 3 small exons. The genes are located close together and in mouse they are found to be less than 10 kb apart (8, 9). Because of the close structural resemblance of the guanylin peptides to ST, as well as the extraordinary high levels of expression of these ligands and their receptor GC-C in the intestinal epithelium, most of the initial research work on them was focused on their effects on the intestine. Gradually, the role of these peptides, especially UGN, in the regulation of fluid electrolyte balance through their action on the kidneys was elucidated. The role played by UGN in the body’s sodium balance can be seen as the mammalian counterpart of the osmoregulatory role of UGN in eurohyaline fish. These fish can adapt to living in salt as well as fresh water. On moving to the sea, they up-regulate the expression of UGN to adapt to the high salt content of sea water (11). This falls perfectly in line with the finding that in mouse, UGN expression increased significantly in the intestine and kidneys with high salt load delivered via drinking water (12). In addition, the 24hr urinary UGN was increased in rats on a high salt diet compared to low salt diet (13). Additional findings that established UGN as a regulator of sodium balance were that UGN, GN, as well as ST stimulated the urinary excretion of sodium, chloride, potassium and water in live animals as well as ex vivo, in isolated perfused kidneys (14-20). It was also shown that UGN and prouroguanylin circulate in the blood of human beings as well as animals, indicating that they might have a hormone like effect (21-24). 3 Based on these findings it was hypothesized that UGN might be the hormone that works in an endocrine axis linking the GI system to the kidneys as depicted in Figure 2 (26). This endocrine axis sends the message to the kidneys to increase sodium excretion in the event of high enteral salt intake. This is similar to the mechanism by which the natriuretic peptides (ANP and BNP) act to excrete the extra salt from the body via the kidneys. In addition, UGN might also act in a local paracrine/autocrine manner involving an intra- renal mechanism (25, 26). Figure 2. Schematic depiction of the hormonal link between the intestine and kidneys through the secretion of UGN by the intestine into the circulation. On ingestion of NaCl, the intestine senses the salt load and secretes UGN which acts on the kidneys to cause natriuresis to excrete the excess salt. Presence of this enterorenal endocrine axis was supported by studies by Lorenz et al (27). Atrial natriuretic protein (ANP), brain natriuretic protein (BNP) and UGN secreted by the cardiac tissue is also believed to participate in the natriuresis seen after high salt loads. The intrarenal paracrine/autocrine effect of the UGN and ANP secreted by the kidneys in the process of natriuresis after oral salt load is not shown in this figure. From Forte et al (26). 4 Perhaps, the most significant and revealing finding comes from the UGN knockout mouse model. To disrupt the UGN gene locus, a targeted allele was generated by using a targeting vector that had 140 bp of the promoter region and 807 bp of exon 1 replaced by a PMC1neo polyA+ cassette in the negative orientation (27).