Angiotensin-converting enzyme-2 (ACE2) enhances the degradation of ANG II and its expression is altered in diabetic kidneys, but the regulation of this enzyme in the urine is unknown. Urinary ACE2 was studied in the db/db model of type 2 diabetes and stretozotocin (STZ)-induced type 1 diabetes during several physiological and pharmacological interventions. ACE2 activity in db/db mice was increased in the serum and to a much greater extent in the urine compared with db/m controls. Neither a specific ANG II blocker, telmisartan, nor an ACE inhibitor, captopril, altered the levels of urinary ACE2 in db/db or db/m control mice. High-salt diet (8%) increased whereas low-salt diet (0.1%) decreased urinary ACE2 activity in the urine of db/db mice. In STZ mice, urinary ACE2 was also increased, and insulin decreased it partly but significantly after several weeks of administration. The increase in urinary ACE2 activity in db/db mice reflected an increase in enzymatically active protein with two bands identified of molecular size at 110 and 75 kDa and was associated with an increase in kidney cortex ACE2 protein at 110 kDa but not at 75 kDa. ACE2 activity was increased in isolated tubular preparations but not in glomeruli from db/db mice. Administration of soluble recombinant ACE2 to db/m and db/db mice resulted in a marked increase in serum ACE2 activity, but no gain in ACE2 activity was detectable in the urine, further demonstrating that urinary ACE2 is of kidney origin. Increased urinary ACE2 was associated with more efficient degradation of exogenous ANG II (10(-9) M) in urine from db/db compared with that from db/m mice. Urinary ACE2 could be a potential biomarker of increased metabolism of ANG II in diabetic kidney disease.

A newly produced murine recombinant ACE2 was characterized in vivo and in vitro. The effects of available ACE2 inhibitors (MLN-4760 and two conformational variants of DX600 –linear and cyclic) were also examined. When murine ACE2 was given to mice for 4 weeks, a marked increase in serum ACE2 activity was sustainable. In acute studies, mouse ACE2 (1mg/kg) obliterated hypertension induced by angiotensin II infusion by rapidly decreasing plasma angiotensin II. These effects were blocked by MLN-4760 but not by either form of DX600. In vitro, conversion from angiotensin II to angiotensin-(1–7) by mouse ACE2 was blocked by MLN-4760 (10−6M) but not by either form of DX600 (10−5M). Quantitative analysis of multiple angiotensin peptides in plasma ex vivo revealed formation of angiotensin-(1–9) from angiotensin I by human but not by mouse ACE2. Both human and mouse ACE2 led to the dissipation of angiotensin II with formation of angiotensin-(1–7). By contrast, mouse ACE2-driven angiotensin-(1–7) formation from angiotensin II was blocked by MLN-4760 but not by either linear or cyclic DX600. In conclusion, sustained elevations in serum ACE2 activity can be accomplished with murine ACE2 administration thereby providing a strategy for ACE2 amplification in chronic studies using rodent models of hypertension and cardiovascular disease. Human, but not mouse ACE2, degrades angiotensin I to form angiotensin-(1–9). There are also species differences regarding rodent and human ACE2 inhibition by known inhibitors such that MLN-4760 inhibits both human and mouse ACE2 whereas DX600 only blocks human ACE2 activity.