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. 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.
