Abstract 242: The pH Influences Angiotensin-(1-7) Forming Enzyme Activities in the Kidney

The kidney plays a pivotal role in acid-base balance. Hydrogen and bicarbonate titration through the nephron produces ranges in urinary pH from 4.5 to 8. All of the components of the renin angiotensin system (RAS) are expressed in the kidney and intrarenal angiotensin II (Ang II) and Ang-(1-7) are formed by multiple independent enzymatic pathways. Most enzymes function optimally at the pH of their native environment. Since small changes in pH can have significant effects on enzyme activities, we investigated the influence of pH on renal Ang II processing to Ang-(1-7). Wild type (WT) and angiotensin converting enzyme 2 (ACE2) knock out (KO) mouse kidney homogenates (20-30 μg protein) were incubated for 30 min at 37°C in 50 mM citrate, phosphate or glycine buffer (pH 4-10) containing 50 μM substrate. First, activity was analyzed using the fluorogenic substrate MCA-Ala-Pro-Lys(Dnp)-OH. A second approach, matrix assisted laser desorption ionization mass spectrometry ((MALDI MS), allowed for the use of the natural substrate, Ang II. In the WT, the fluorogenic assay system detected activity at pH 4, 5, 5.5, 6, 7, 8, 9, and 10 with minimal differences. Using MALDI MS, renal enzymatic Ang-(1-7) formation was observed at a pH range between 4 and 9 with two peaks at pH 5.5 and 8. At pH