A method to evaluate the renin-angiotensin system in rat renal cortex using a microdialysis technique combined with HPLC-fluorescence detection.
A microdialysis (MD) technique, combined with HPLC-fluorescence (FL) detection, was developed for the evaluation of the tissue-specific renin-angiotensin system (RAS) in the rat renal cortex. An MD probe constructed with a hydrophilic hollow fiber dialysis tubing, AN69, showed high recovery (more than 50%) in vitro for all four angiotensins: angiotensin I (Ang I), Ang II, Ang III, and Ang (1-7). Angiotensins, successfully derivatized with m-BS-ABD-F, a water-soluble fluorogenic reagent that has a 2,1,3-benzoxadiazole (benzofurazan) structure, could be simultaneously determined by coupled-column HPLC. The detection limit for Ang I, Ang II, Ang III, and Ang (1-7) were 94, 44, 47, and 83 fmol, respectively. All these peptides were determined with good linearity (0.0125-3.1 microM, equivalent to 0.25-62 pmol, correlation coefficient >0.99) and good precision (recovery >91%). In the MD studies, generation of Ang (1-7) and Ang II was observed when Ang I was perfused, and Ang (1-7) was the major biologically active angiotensin found in the dialysate samples. The concentration of Ang (1-7) and Ang II in the dialysate samples showed good correlation to that of Ang I in a MD perfusate (20-100 microM). Cleavage of Ang I to Ang (1-7) was drastically suppressed by the co-perfusion of phoshoramidon (0.5-5 mM), an inhibitor of neprilysin, which generates Ang (1-7) from Ang I. These results are consistent with the previously reported characteristics of tissue-specific renal RAS, suggesting that our MD/HPLC-FL system may have the potential to be employed to evaluate tissue-specific RAS in the rat renal cortex.
Kajiro
Toshi
T
Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan.
Nakajima
Yuki
Y
Fukushima
Takeshi
T
Imai
Kazuhiro
K
D000809:Angiotensins / Q000032:analysis*; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D003956:Dialysis; D004867:Equipment Design; D005456:Fluorescent Dyes; D007672:Kidney Cortex / Q000737:chemistry*; D008832:Microchemistry; D051381:Rats; D012084:Renin-Angiotensin System
D000809:Angiotensins; D005456:Fluorescent Dyes