Sex differences in renal angiotensin converting enzyme 2 (ACE2) activity are 17β-oestradiol-dependent and sex chromosome-independent - PubMed (original) (raw)
Sex differences in renal angiotensin converting enzyme 2 (ACE2) activity are 17β-oestradiol-dependent and sex chromosome-independent
Jun Liu et al. Biol Sex Differ. 2010.
Abstract
Background: Angotensin converting enzyme 2 (ACE2) is a newly discovered monocarboxypeptidase that counteracts the vasoconstrictor effects of angiotensin II (Ang II) by converting Ang II to Ang-(1-7) in the kidney and other tissues.
Methods: ACE2 activity from renal homogenates was investigated by using the fluorogenic peptide substrate Mca-YVADAPK(Dnp)-OH, where Mca is (7-methoxycoumarin-4-yl)-acetyl and Dnp is 2,4-dinitrophenyl.
Results: We found that ACE2 activity expressed in relative fluorescence units (RFU) in the MF1 mouse is higher in the male (M) compared to the female (F) kidney [ACE2 (RFU/min/μg protein): M 18.1 ± 1.0 versus F 11.1 ± 0.39; P < 0.0001; n = 6]. Substrate concentration curves revealed that the higher ACE2 activity in the male was due to increased ACE2 enzyme velocity (Vmax) rather than increased substrate affinity (Km). We used the four core genotypes mouse model in which gonadal sex (ovaries versus testes) is separated from the sex chromosome complement enabling comparisons among XX and XY gonadal females and XX and XY gonadal males. Renal ACE2 activity was greater in the male than the female kidney, regardless of the sex chromosome complement [ACE2 (RFU/min/μg protein): intact-XX-F, 7.59 ± 0.37; intact-XY-F, 7.43 ± 0.53; intact-XX-M, 12.1 ± 0.62; intact-XY-M, 12.7 ± 1.5; n = 4-6/group; P < 0.0001, F versus M, by two-way ANOVA]. Enzyme activity was increased in gonadectomized (GDX) female mice regardless of the sex chromosome complement whereas no effect of gonadectomy was observed in the males [ACE2 (RFU/min/μg protein): GDX-XX-F, 12.4 ± 1.2; GDX-XY-F, 11.1 ± 0.76; GDX-XX-M, 13.2 ± 0.97; GDX-XY-M, 11.6 ± 0.81; n = 6/group]. 17β-oestradiol (E2) treatment of GDX mice resulted in ACE2 activity that was only 40% of the activity found in the GDX mice, regardless of their being male or female, and was independent of the sex chromosome complement [ACE2 (RFU/min/μg protein): GDX+E2-XX-F, 5.56 ± 1.0; GDX+E2-XY-F, 4.60 ± 0.52; GDX+E2-XX-M, 5.35 ± 0.70; GDX+E2-XY-M, 5.12 ± 0.47; n = 6/group].
Conclusions: Our findings suggest sex differences in renal ACE2 activity in intact mice are due, at least in part, to the presence of E2 in the ovarian hormone milieu and not to the testicular milieu or to differences in sex chromosome dosage (2X versus 1X; 0Y versus 1Y). E2 regulation of renal ACE2 has particular implications for women across their life span since this hormone changes radically during puberty, pregnancy and menopause.
Figures
Figure 1
Synthetic and catabolic pathways of Ang II.
Figure 2
Dependence of renal angiotensin converting enzyme 2 (ACE2) activity on pH in the MF1 mouse. (A) Effect of pH on the time course of product formation in the female mouse. Data are representative of three experiments performed in triplicate. (B) Effect of pH on enzyme product formation at 150 min; *P < 0.05 and **P < 0.01 versus pH 7.5, n = 4/group.
Figure 3
Dependence of renal angiotensin converting enzyme 2 (ACE2) activity on protein concentration in the MF1 mouse. Effect of protein concentration on the time course of product formation in (A) male and (B) female mice. Data are representative of three experiments performed in triplicate. (C) Effect of protein concentration on product formation at 40 min in male and female mice; *P < 0.01 versus female by _t_-test; two-way ANOVA revealed a significant effect of protein (P < 0.0001) and sex (P < 0.0001) on renal ACE2 activity; n = 4/group.
Figure 4
Dependence of renal ACE2 activity on substrate concentration in the MF1 mouse. Effect of substrate concentration on the time course of product formation in (A) male and (B) female mice. Data are representative of three experiments performed in triplicate. (C) Effect of substrate concentration on product formation at 40 min in male and female mice. Also shown is the determination of _K_m and _V_max; *P < 0.001 versus female by _t_-test; n = 4-5/group. Two-way ANOVA revealed a significant effect of substrate (P < 0.0001) and sex (P < 0.0001) on renal ACE2 activity.
Figure 5
Angiotensin converting enzyme 2 (ACE2) activity in the kidney, heart and lung of the MF1 mouse. Comparison of ACE2 activity in male and female mice tissue homogenates using 10 μg kidney and 40 μg heart and lung protein and 30 μM substrate concentration for all three tissues; *P < 0.001 versus female by _t_-test; n = 5/group.
Figure 6
Angiotensin converting enzyme 2 (ACE2) protein and messenger RNA (mRNA) expression in the MF1 mouse kidney. (A) Quantitation of ACE2 protein expression from Western blots (upper) of male and female mouse renal homogenates; *P < 0.05 versus female; n = 7/group. (B) Quantitation of renal ACE2 mRNA expression determined by real-time polymerase chain reaction in male (n = 5) and female (n = 7) mouse kidney; *P < 0.05 versus female.
Figure 7
Renal angiotensin converting enzyme 2 (ACE2) activity in the four core genotypes (FCG) in the intact and GDX state treated with and without oestradiol (E2). Shown are the mean ± standard error of mean for renal ACE2 activity in gonadally intact (open bars), GDX (striped bars) and E2 treated GDX (hatched bars) FCG mice; n = 6/group. ANOVA analyses revealed a significant effect of sex (P < 0.001, *intact-female versus intact-male), gonadectomy in the female (P < 0.001, #intact-female versus GDX-F) and E2 treatment in all GDX mice (P < 0.001, ‡GDX+E2 versus GDX) on renal ACE2 activity (see Results for details).
References
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