Response to Method of Blood Collection May Explain the Suppression of Plasma Renin Concentration in Prorenin Transgenic Mice
In responding to the letter by Campbell, 1 it is important to point out that this issue has no bearing on the major finding of our article, 2 that elevations in plasma prorenin alone do not result in cardiac or renal pathologies. Nevertheless, Campbell 1 brings up an important issue, that measurement of plasma renin concentration (PRC) is fraught with potential artifacts. In addition to the ability of stress and anesthesia to cause an acute release of renin, the mouse is particularly sensitive
... icularly sensitive to blood pressure decreases resulting from sampling too much blood because of its small blood volume compared with rats and humans. We are aware of all of these factors and have developed sampling and assay techniques that are reproducible and reflect the expected physiological modulation of PRC. We apologize for not including these details in our most recent article, and they are described below. Mice are lightly anesthetized with ether, and blood is collected from the retro-orbital sinus with a short heparinized microhematocrit capillary tube. No more than 3 drops of blood are collected into a microcentrifuge tube containing 10 L of 100-mmol/L EDTA (pH 7.4). Every effort is made to perform the blood collection swiftly and without stress to the animal. Plasma is recovered and immediately frozen at Ϫ80°C until the assay is performed. On the day of the assay we thaw the sample on the bench and perform the angiotensin I generation assay in the presence of EDTA, PMSF (inactivates trypsin and other serine proteases), and 8-hydroxyquinoline (inhibits breakdown of angiotensin peptides). Although it is very difficult to prove that we have no artifact in our renin measurements, we have found that this method gives lower renin concentrations in control mice than other sampling methods that we have tried (saphenous vein, cheek pouch, etc). In addition, our TTR-mProren-7 mice develop an angiotensin II-dependent hypertension ( Figure 5 of the article 2 ), and their PRCs drop, as would be expected, because both pressure and angiotensin II are known to suppress renin release from the kidney. Moreover, when we place mice on pharmacological inhibitors of the renin-angiotensin system (eg, angiotensin-converting enzyme inhibitors) for as little as 5 days, their PRCs increase up to 15-fold (our unpublished results), as would be expected when removing angiotensin II feedback suppression. Thus, our assay reflects the expected physiological modulation of PRC. In conclusion, we agree with Campbell 1 that animal handling, anesthesia, and assay methods can affect PRC measurements, but we remain convinced that the conditions that we use reflect the normal and expected regulation of kidney renin by blood pressure and angiotensin II, and the results do not justify the suspicion of an assay artifact. Disclosures None.