The renin-angiotensin system (RAS) is a phylogenetically well-preserved system in control of body fluid homeostasis and blood pressure as well as growth and proliferation. Studies in knockout mice suggest an important role of the RAS in the development of the kidney and ureteral tract. Studies from non-mammalian organisms can contribute in several ways. In addition to providing insight into the evolution of this system, certain models systems provide methodological advantages. We have used two non-mammalian systems, the zebrafish (Danio rerio) and the elasmobranch, dogish, (Squalus acanthias) The dogfish shark rectal salt gland has been extensively exploited to study epithelial transport. Studies on the dog fish were completed in FY01 and reported in the current year. See bibliography. Studies in the current year concentrated on zebrafish. The transparency of the developing embryo, the ability to perform genetic manipulations and the availability of genomic tools make the zebrafish a valuable model for studies of vertebrate biology. Studies in zebrafish might potentially contribute to elucidating the mechanisms causing cell-specific expression of renin and the contribution of the renin-angiotensin system to kidney development. Identification of renin and of other components of the RAS at the molecular level would seem to be a prerequisite for such studies. Since renin has only been cloned in mammalians we performed experiments to establish and characterize the RAS in teleost fish. By screening a zebrafish kidney cDNA library using a human renin cDNA probe, several identical clones were obtained that had an about 80% homology with cathepsin D (zCATD), but only a low homology with renin sequences. ZCATD was expressed at highest levels in the kidney. Furthermore, we cloned and characterized the first teleost angiotensinogen cDNA (zAgt) and found it expressed in liver and kidney. Using a 17 amino acid teleost-specific synthetic substrate and protein separation by capillary electrophoresis, zCATD expressed in COS 7 cells was found to be an angiotensin-generating enzyme with an acid pH optimum. In order to study renin like activity in fish, we are currently undertaking expression studies of zebrafish angiotensinogen. To date we have established that zebrafish kidney extracts and goldfish plasma generate angiotensin at acid pH. Our results suggest that another aspartyl protease, possibly cathepsin D, may be responsible for enzymatic formation of angiotensin in the kidney of zebrafish and other teleosts, findings with implications for the evolution of lysosomal aspartic proteases and of the renin angiotensin system. The availability of genomic sequence information about both pufferfish and zebrafish has allowed further assessment of the aspartyl proteases that might function to generate renin from angiotensinogen. Using bioinformatic approaches two additional renin-like aspartyl protease sequences have been identified in both teleost species. Subsequent studies will examine their expression patterns, and if the expression pattern is consistent with a renin-like role, we will examine their enzymatic properties. In mammalian species, renin secretion is sensitive to NaCl concentration in the distal nephron, an effect mediated through the SLC12A1 transporter. A new direction in the past year has been to identify the teleost homologs of this cotransporter, in order to determine if these anatomic relationships are conserved. In mammals there are three alternate splice variants of the SLC12A1 co-transporter, with highly specific expression patterns. Informatics strategies have established that two of these appear to be present in the teleost, and further studies will examine their distribtuion and potential role. We have proceeded to express zebrafish angiotensinogen using a bacillovirus expression system. The goal of these experiments is to test whether the full length substrate might confer a different pH optimum on the reaction to generate angiotensin I. The full length protein has been expressed, and the pH optimum experiments are underway.
