Rhace2

Serum was used to measure blood urea nitrogen, creatinine, sodium, potassium, and chloride concentrations. Plasma was stored at −80°C until batched measurement of equilibrium concentrations of APs, including AT1, AT2, Ang1‐9, Ang1‐7, Ang1‐5, angiotensin 3 (AT3), and angiotensin 4 (AT4), was performed as previously described.¹⁸, ¹⁹, ²⁰, ²², ²⁶ Briefly, plasma was spiked with stable isotope‐labeled standards for each AP, allowed to reach equilibrium at 37°C, and assayed using liquid chromatography‐tandem mass spectrometry (Attoquant Diagnostics, Vienna, Austria). Use of equilibrium assay avoids the need for special collection and handling requirements at the time of blood collection, increases the signal‐to‐noise ratio of the assay, and results in AP concentrations higher than, but proportional to plasma concentrations using enzyme inhibitors at the time of blood collection.¹⁸, ²⁷ To assess the effect of exogenous rhACE2 on the relative concentrations of APs, plasma samples were incubated in vitro with 5 μg/mL of rhACE2 (R&D Systems, Minneapolis, Minnesota) at 37°C, followed by assay of equilibrium concentrations of AT1, AT2, Ang1‐9, Ang1‐7, and Ang1‐5 using previously described methods.¹⁸, ¹⁹

Serum was used to measure BUN, creatinine, sodium, potassium, and chloride. Lithium heparin plasma was stored at −80°C until batched measurement of equilibrium concentrations of APs, including AT1, AT2, Ang1‐9, Ang1‐7, Ang1‐5, angiotensin 3 (AT3), and angiotensin 4 (AT4) was performed as previously described.¹, ¹⁶, ¹⁷, ²², ²³ Briefly, plasma was spiked with stable isotope‐labeled standards for each AP, allowed to reach equilibrium at 37°C, and assayed using liquid chromatography‐tandem mass spectrometry (Attoquant Diagnostics, Vienna, Austria). Use of equilibrium assays avoids the need for special collection and handling requirements at the time of blood collection, increases the signal‐to‐noise ratio of the assay, and results in AP concentrations higher than, but proportional to, plasma concentrations using enzyme inhibitors at time of blood collection.¹⁶, ²⁴ To assess the effect of exogenous ACE2 on the relative concentrations of selected APs, plasma from cats with CM was assayed after in vitro incubation with 5 μg/mL of rhACE2 (R&D Systems, Minneapolis, Minnesota) at 37°C, followed by measurement of equilibrium concentrations of AT1, AT2, Ang1‐9, Ang1‐7, and Ang1‐5.¹⁶, ¹⁷