Response to "Determination of Sex Differences in Activities of Angiotensin-Converting Enzyme 2 (ACE2) Requires an Activity Assay That Doesn't Underestimate ACE2"

To the Editor: We are writing with reference to the letter “Determination of Sex Differences in Activities of Angiotensin-Converting Enzyme 2 (ACE2) Requires an Activity Assay That Doesn’t Under estimate ACE2” from Pedersen and Lazartigues.1 We appreciate the comments of the authors on our recent article examining enzymatic activity of key enzymes in the renin-angiotensin system that impact angiotensin (Ang) II and Ang (1–7) levels in male and female WKY rats.2 Pedersen and Lazartigues offer concern that the methodology used to measure ACE2 activity in our manuscript underestimates activity and may mask a potential sex difference. This is a valid concern; however, we will be happy to better explain the troubleshooting that was done in our laboratory to optimize our results. We standardized the ACE2 activity assay in our tissues of interest based on work from Dr Lazartigues’ group.3 Preliminary studies in our laboratory measured ACE2 activity in the presence of 2 different amounts of protein homogenate (40 and 100 μg) and at 3 different concentrations of the ACE2 inhibitor DX600 (1, 2, and 10 μM) in renal cortex samples from male WKY rats. We did not find significant differences in ACE2 activity using 40 μg of tissue homogenate in the presence of either 1 μM DX600 (15±3 RFU/min/mg protein) or 10 μM DX600 (13±1 RFU/min/mg protein). Based on this result, all future studies were performed with the lower concentration of DX600. Additional studies showed that we obtained the most reliable and consistent results using a greater amount of protein, and for the article all of the data were obtained using 100 μg of tissue homogenate. All troubleshooting of the assay in our laboratory was done using tissue from male WKY rats; therefore, we cannot attest to potential sex differences that may exist at higher concentrations of DX600 and certainly yield that using alternative methods of assessment may find a sex differences in renal ACE2 activity in SHR. We would propose, however, that this does not detract from the primary novel finding of our study, which was that female WKY rats have greater renal cortical ACE1 activity and greater Ang (1–7) levels than males.2 Moreover, inhibition of ACE1 abolished the sex difference in Ang (1–7), supporting a key role for ACE in regulating renal cortical Ang (1–7) levels in normotensive females. We acknowledged in the article that ACE2 likely contributes to Ang (1–7) levels in the kidney, but it did not appear in normotensive WKY rats to contribute to the sex difference in Ang (1–7). That said, we are interested to further pursue this question using the revised methods suggested by Pedersen and Lazartigues.