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. Furthermore, 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 methodlogical 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. We have just completed studies cloning and characterizing the cyclooxygenase of the rectal salt gland of the dogfish. It shows molecular properties indicating that it is an intermediate form, between COX1 and COX2. Transport effects of COX inhibition provided functional corrleates of the molecular studies. This work has been submitted for publication. With regard to the 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. Likewise, zebrafish kidney extracts and goldfish plasma generated angiotensin predominantly at acid pH. Our results suggest that another aspartyl protease, 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.
