Characterization of ACE and ACE2 Expression within Different Organs of the NOD Mouse

Abstract

Renin angiotensin system (RAS) is known to play a key role in several diseases such as diabetes, and renal and cardiovascular pathologies. Its blockade has been demonstrated to delay chronic kidney disease progression and cardiovascular damage in diabetic patients. In this sense, since local RAS has been described, the aim of this study is to characterize angiotensin converting enzyme (ACE) and ACE2 activities, as well as protein expression, in several tissues of the non-obese diabetic (NOD) mice model. After 21 or 40 days of diabetes onset, mouse serums and tissues were analyzed for ACE and ACE2 enzyme activities and protein expression. ACE and ACE2 enzyme activities were detected in different tissues. Their expressions vary depending on the studied tissue. Thus, whereas ACE activity was highly expressed in lungs, ACE2 activity was highly expressed in pancreas among the studied tissues. Interestingly, we also observed that diabetes up-regulates ACE mainly in serum, lung, heart, and liver, and ACE2 mainly in serum, liver, and pancreas. In conclusion, we found a marked serum and pulmonary alteration in ACE activity of diabetic mice, suggesting a common regulation. The increase of ACE2 activity within the circulation in diabetic mice may be ascribed to a compensatory mechanism of RAS.

Keywords: angiotensin converting enzyme 2; diabetes; renin angiotensin system.

Figure 1

Implementation of angiotensin converting enzyme (ACE) activity assays with different buffers and in different tissues. (a) ACE activity with borate (BB) and phosphate buffer (PB) after increasing amounts (ng) of human recombinant ACE (hrACE). ACE activity at lower concentrations of hrACE was only detected by the use of BB; (b) ACE activity in lung measured with BB (black rhombus), and (c) ACE activity in heart (blue triangle), pancreas (green circle), and liver (red cross) from control (dashed lines) and diabetic (continuous lines) mice. Increasing amounts of micrograms of total protein extracts were tested.

Figure 2

ACE2 activity assays in different tissues and with increasing protein amounts. ACE2 activity was measured in lung (black rhombus), heart (blue triangle), pancreas (green circle), and liver (red cross) from control animals (CONT) (left panel) and diabetic animals (DB) (right panel) mice. Increasing amounts of micrograms of total protein extracts from each studied tissue were tested (1, 3, 5, 7.5, and 10 μg of protein).

Figure 3

ACE activity in serum and different tissues. (a) ACE activity in serum samples from CONT (open bars), DB (closed bars), and insulin-treated diabetic (DB + INS, grey bars) mice at 21 and 40 days after diabetes onset; (b) ACE activity in lungs from CONT, DB, and DB + INS mice; (c) ACE activity in heart, liver, and pancreas from CONT, DB, and DB + INS. * p ≤ 0.05 CONT vs DB.

Figure 4

ACE2 activity in serum and different tissues. (a) ACE2 activity in serum samples from CONT (open bars), DB (closed bars), and DB + INS (grey bars) mice at 21 and 40 days after diabetes onset; (b) ACE2 activity in lung, heart, liver, and pancreas from CONT, DB, and DB + INS. * p ≤ 0.05 CONT vs. DB; ^# p ≤ 0.05 DB vs. DB + INS; ^$ p ≤ 0.05 21 vs. 40 days of follow up.

Figure 5

ACE2/ACE activity ratio in serum and different tissues. (a) ACE2/ACE activities ratio in serum samples from CONT (open bars), DB (closed bars), and DB + INS (grey bars) mice at 21 and 40 days after diabetes onset; (b) ACE2/ACE activity ratio in lungs from CONT, DB, and DB + INS mice; (c) ACE2/ACE activity ratio in heart, liver, and pancreas from CONT, DB, and DB + INS. * p ≤ 0.05 CONT vs. DB; ^# p ≤ 0.05 DB vs. DB+INS; ^$ p ≤ 0.05 21 vs. 40 days of follow up.

Figure 6

ACE and ACE2 protein expression in different tissues. Upper panel: normalized ACE and ACE2 protein expression in lung (a); heart (b); liver (c); and pancreas (d) homogenates from CONT (open bars), DB (closed bars), and DB + INS (grey bars) mice at 21 and 40 days after diabetes onset. Lower panel: representative images depicting bands corresponding to ACE and ACE2 and protein controls. * p ≤ 0.05 CONT vs. DB; ^# p ≤ 0.05 DB vs. DB + INS; ^$ p ≤ 0.05 21 vs. 40 days of follow up.