Regulation of Renal Angiotensin Converting Enzyme 2 Activity by Angiotensin IIInduced Hypertension

1
Regulation of Renal Angiotensin Converting Enzyme
2 Activity by Angiotensin II-Induced
Hypertension and Gonadal Sex Anita Mazloom, Jun
Liu, Wei Zheng, Hong Ji, Xie Wu and Kathryn
Sandberg Division of Nephrology and
Hypertension, Department of Medicine2 Georgetown
University Medical Center, Washington, DC
Georgetown University
For most of their lives, women have lower blood
pressure and a lower incidence of hypertension
than men of the same age. While this is a widely
observed phenomenon throughout the world
regardless of race and ethnicity, the mechanisms
underlying this sex difference remain poorly
understood. The renin angiotensin system (RAS)
plays a critical role in the control of blood
pressure and inhibitors of angiotensin II (Ang
II) synthesis are widely used clinically to treat
the vasoconstriction associated with
hypertension. Furthermore, we and others have
shown that sex differences exist in the
regulation of the RAS and that females have lower
blood pressure in response to Ang II infusion
than males. Angiotensin converting enzyme II
(ACE2) is a newly discovered enzyme in the RAS
cascade that counteracts the vasoconstrictor
effects of Ang II by metabolizing this hormone to
the vasodilator, Ang-(1-7). In this study, we
investigated the effect of gonadal sex on renal
ACE2 activity in a mouse model of Ang II-induced
hypertension to determine if sex differences in
ACE2 activity could contribute to why females
have a lower incidence of RAS-dependent
hypertension than males. Objectives 1) To
develop an assay for measuring ACE2 activity in
the mouse renal cortex 2) To determine the
effect of gonadal sex on renal ACE2 activity
under normotensive conditions and, 3) To
determine the effect of gonadal sex on renal ACE2
activity after Ang II-induced hypertension.
Results Under normotensive conditions, the
initial rate of ACE2 activity (t1/2) was faster
in the female kidney than in the male while the
maximum enzyme velocity (Vmax) was equivalent in
both sexes. After Ang II-induced hypertension,
both the Vmax was increased in both sexes
however, the magnitude of the increase was
greater in the male. Conclusions The increase in
ACE2 Vmax observed in both sexes after Ang II
infusion suggests the activity of renal ACE2 is
up-regulated to counteract the hypertensive
effects of Ang II. Furthermore, sex differences
in the regulation of renal ACE2 activity in the
normotensive and hypertensive states suggests
that differential ACE2 regulation contributes to
sex differences in blood pressure control and
RAS-dependent hypertension.  
Pre-menopausal women are protected against
hypertension and blood pressure compared to
age-matched men.1 These sex differences have
been proposed to be linked to activity in the
renin angiotensin system (RAS). Patients who
have high activity in this system are at risk for
hypertension. Furthermore, ACE inhibitors and
Angiotensin II type I receptor antagonists are
effective drugs for controlling hypertension. In
the Renin Angiotensin system, Renin released from
the kidney cleaves Angiotensinogen into
Angiotensin I (Ang I). Angiotensin converting
enzyme (ACE) then acts on Angiotensin I cleaving
it into Angiotensin II (Ang II). Ang II has
various effects in the body. It can trigger the
release of Aldosterone from the adrenal cortex
leading to sodium and fluid retention and
increased blood volume and also can directly
cause systemic vasoconstriction. A recently
discovered enzyme, ACE2, hydrolyzes Ang I into
Angiotensin (1-9) and can also cleave Ang II into
Angiotensin (1-7). The major biologically active
product of ACE2 is Ang (1-7). ACE2 has a role in
balancing the vasoconstriction and other effects
of AngII with Ang(1-7) which have vasodilatory
effects.2 Based on this system, we have examined
the sex differences between male and female mice
models by measuring ACE2 activity.
2) Determined effect of gonadal sex on renal ACE2
activity under normotensive conditions
1) Developed an assay for measuring ACE2 activity
in mouse renal cortex optimized conditions
including substrate concentration, protein dose,
temperature, and time course no previous assay
has been developed
3) Determined effect of gonadal sex on renal ACE2
activity after AngII-induced hypertension
previously, males have higher blood pressure in
models of hypertension

•  
• Preparation of Protein Samples Weigh tissue
  samples and homogenize samples with lysis buffer
  remove supernatant after centrifugation and store
  at -70 degrees
• BSA Assay Prepare dilutions of BSA standard,
  dilute tissue samples (50x), prepare 96 well
  plate Add 10 ?L standard/samples to each well,
  add 100 ?L Bio-Rad protein dye to each well and
  incubate for 5 minutes at RT, run plate reader
• ACE2 Assay Prepare dilutions of Captopril/MLN
  (ACE and ACE2 inhibitors respectively) and
  fluorescence substrate stock solutions, prepare
  96 well plate Add each of the following in the
  order given and then run plate reader Victor 3.0
  1. Reaction Buffer add volume so total 100
  ?L 2. Captopril (10 ?M), 3. MLN (10 ?M or 0 for
  sample with only one inhibitor), 4. Substrate (25
  ?M), 5. Sample (protein concentration 40 ?g/ml)
  add calculated volume from concentration found in
  BSA assay

The increase in ACE2 Vmax that was observed in
both sexes after treatment with Ang II suggests
that the activity of renal ACE2 is up-regulated
to offset the hypertensive effects of Ang II. In
addition, the sex differences in the regulation
of renal ACE2 activity in both the basal and
hypertensive states suggests that differential
ACE2 regulation contributes to sex differences in
blood pressure control and RAS-dependent
hypertension.

• Chen, Y.F. Sexual dimorphism of hypertension.
  Curr Opin Nephrol Hypertens 5, 181-5 (1996).
• Raizada MK, Ferreira AJ. ACE2 A New Target for
  Cardiovascular Disease Therapeutics. J Cardiovas.
  Pharmacol. Aug, 2007. Vol 50(2) 112-9

This work is supported by NIH R01 HL57502 and R01
HL19291
Spring 2008